Wet granulation formulation of a growth hormone secretagogue

ABSTRACT

The present invention relates to a pharmaceutical composition and a process for the preparation of a tablet containing a growth hormone secretagogue as the active ingredient. The tablet is prepared by forming a powder blend of the active ingredient N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4&#39;-piperdin]-1&#39;-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methyl-propanamide, or a pharmaceutically acceptable salt thereof, in particular the methanesulfonate salt, with a binder/diluent, a first diluent, a second diluent, a first portion of a disintegrant, and a lubricant; wet granulating the powder blend with a solution of ethanol/water to form granules; drying the granules to remove the ethanol/water; adding a second portion of a disintegrant; lubricating the granules; and compressing the dried granules into the desired tablet form. The present invention further relates to a novel amorphous form of the compound N-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4&#39;-piperdin]-1&#39;-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide methanesulfonate which is produced directly as a result of the process of tablet formulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from PCT Application No.PCT/US96/17196, filed Oct. 23, 1996, which claims the benefit of U.S.Ser. No. 60/005,897, filed Oct. 27, 1995 and U.S. Ser. No. 60/005,901,filed Oct. 27, 1995.

FIELD OF THE INVENTION

The present invention relates to solid dosage formulations comprising agrowth hormone secretagogue and process for their manufacture. Morespecifically, the invention relates to a wet granulation formulation ofa growth hormone secretagogue compound. The present invention furtherrelates to an amorphous form of a growth hormone secretagogue, processesfor its preparation and uses thereof.

BACKGROUND OF THE INVENTION

Growth hormone, which is secreted from the pituitary, stimulates growthof all tissues of the body that are capable of growing. In addition,growth hormone is known to have the following basic effects on themetabolic processes of the body: (1) Increased rate of protein synthesisin all cells of the body; (2) Decreased rate of carbohydrate utilizationin cells of the body; (3) Increased mobilization of free fatty acids anduse of fatty acids for energy. A deficiency in growth hormone secretioncan result in various medical disorders, such as dwarfism.

Various ways are known to release growth hormone. For example, chemicalssuch as arginine, L-3,4dihydroxyphenylalanine (L-DOPA), glucagon,vasopressin, and insulin induced hypoglycemia, as well as activitiessuch as sleep and exercise, indirectly cause growth hormone to bereleased from the pituitary by acting in some fashion on thehypothalamus perhaps either to decrease somatostatin secretion or toincrease the secretion of the known secretagogue growth hormonereleasing factor (GRF) or an unknown endogenous growth hormone-releasinghormone or all of these.

In cases where increased levels of growth hormone were desired, theproblem was generally solved by providing exogenous growth hormone or byadministering GRF or a peptidal compound which stimulated growth hormoneproduction and/or release. In either case the peptidyl nature of thecompound necessitated that it be administered by injection. Initiallythe source of growth hormone was the extraction of the pituitary glandsof cadavers. This resulted in a very expensive product and carried withit the risk that a disease associated with the source of the pituitarygland could be transmitted to the recipient of the growth hormone.Recombinant growth hormone has become available which, while no longercarrying any risk of disease transmission, is still a very expensiveproduct which must be given by injection or by a nasal spray.

Other compounds have been developed which stimulate the release ofendogenous growth hormone such as analogous peptidyl compounds relatedto GRF or the peptides of U.S. Pat. No. 4,411,890. These peptides, whileconsiderably smaller than growth hormones are still susceptible tovarious proteases. As with most peptides, their potential for oralbioavailability is low. Non peptidal growth hormone secretagogues with abenzolactam structure are disclosed e.g., in U.S. Pat. Nos. 5,206,235,5,283,241, 5,284,841, 5,310,737, 5,317,017, 5,374,721, 5,430,144,5,434,261, 5,438,136 and PCT Publications WO 95/03289, WO 95/03290, WO95/09633. Other growth hormone secretagogues are disclosed in PCT PatentPublications WO 94/11012, WO 94/13696, WO 94/19367, WO 95/13069 and WO95/14666.

In particular, Examples 18, 19 and 55 of U.S. Pat. No. 5,536,716 (PCTPatent Publication WO 94/13696) and Proc. Natl. Acad. Sci. USA, 92,7001-7005 (July 1995) disclose the compoundN-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamide,and salts thereof, especially the methanesulfonate salt, which has thestructure: ##STR1##

This compound is a growth hormone secretagogue which stimulates therelease of growth hormone in humans and animals. This property can beutilized to promote the growth of food animals to render the productionof edible meat products more efficient, and in humans, to treatphysiological or medical conditions characterized by a deficiency ingrowth hormone secretion, and to treat medical conditions which areimproved by the anabolic effects of growth hormone.

U.S. Pat. No. 5,536,716 and PCT Patent Publication WO 94/13696 disclosemethods for preparing this compound (see Examples 18, 19 and 55). Inparticular, Example 55 states that the compound prepared byrecrystallization from ethyl acetate-ethanol-water had a melting pointof 166-168° C. Proc. Natl. Acad. Sci. USA, 92, 7001-7005 (July 1995)notes that this compound isolated as a monohydrate had a melting pointof 168-170° C.

Standard methods for tablet formulation of the active ingredient such asdirect compression suffer from problems. In particular, this compound isrelatively unstable in standard pharmaceutical formulations. Inaddition, this compound as a bulk drug suffers from poor flowproperties, nevertheless, wet granulation was discovered to overcomethese difficulties preparing tablet formulations. Tablets prepared bythe wet granulation method produced excellent content uniformity,coupled with suitable tablet dissolution and stability. The tablets ofthe present invention, prepared by wet granulation, possessed goodhardness at normal machine pressures.

The present invention is also concerned with pharmaceutical formulationsprepared by the subject process and their use in the treatment ofcertain disorders and diseases.

Exemplifying the present invention is the process comprising the stepsof:

(1) forming a powder blend of the active ingredient with abinder/diluent, a first diluent, a second diluent, and a disintegrant,from 2 to 25 minutes using a mixer;

(2) wet granulating the powder blend by adding a solution ofethanol/water to the powder blend while mixing over a 1 to 30 minuteperiod to form granules;

(3) drying the granules to remove the ethanol/water with heated air in afluid bed dryer or tray dryer for 10 minutes to 24 hours;

(4) milling the dried granules to a uniform size;

(5) adding and blending a disintegrant with the dried milled particlesfor 2 to 30 minutes;

(6) adding and blending a lubricant to the mixture containing thedisintegrant for 30 seconds to 20 minutes; and

(7) compressing the lubricated granules mixture into a desired tabletform.

Further illustrating the invention is the process wherein the activeingredient isN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl)-2-amino-2-methylpropanamidemethanesulfonate.

Additional illustrations of the invention include the process wherein:the binder/diluent is pregelatinized starch; the first diluent ismicrocrystalline cellulose; the second diluent is calcium phosphatedibasic; the disintegrant is croscarmellose sodium; and the lubricant ismagnesium stearate. Preferably, the solution of ethanol/water is in therange of 0% to 80% ethanol in water (w/w), more preferably in the rangeof 5% to 75% ethanol/water (w/w) and even more preferably approximately25% ethanol/75% water (w/w).

A further illustration of the invention is the foregoing process furthercomprising the step of applying a coating to the tablet. Moreparticularly illustrating the invention is the process wherein coatingthe tablet is accomplished by:

(1) dry blending titanium dioxide (optionally mixed with talc) withhydroxypropyl methylcellulose and hydroxypropyl cellulose to form a drypowdered blend;

(2) adding the dry powdered blend to water to form a slurry;

(3) adding water to the slurry with stirring to form a suspension; and

(4) applying the suspension to the tablets.

More specifically exemplifying the invention is the process comprisingthe steps of:

(1) forming a powder blend of the active ingredient with pregelatinizedstarch, microcrystalline cellulose, calcium phosphate dibasic, andcroscarmellose sodium, in a mixer for about 3 to 25 minutes;

(2) wet granulating the powder blend by adding a solution of 25%ethanol/75% water (w/w) to the powder blend while mixing over a 1 to 30minute period to form granules;

(3) drying the granules on a tray dryer or a fluid bed dryer for about 1to 12 hours to remove the ethanol/water;

(4) milling the dried granules to a uniform size using a Quadro Comillor Fitz type mill;

(5) adding and blending croscarmellose sodium with the dried milledparticles for about 5 to 30 minutes;

(6) adding and blending magnesium stearate to the mixture containing thecroscarmellose sodium with a V blender for about 1 to 5 minutes; and

(7) compressing the lubricated granules mixture into a desired tabletform.

Another example of the invention is the process wherein the activeingredient isN-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)-ethyl]-2-amino-2-methylpropanamidemethanesulfonate.

In a subclass is the foregoing process further comprising the step ofcoating the tablet. Further exemplifying the invention is the foregoingprocess further comprising the steps of:

(1) dry blending titanium dioxide (optionally mixed with talc) withhydroxypropyl methylcellulose and hydroxypropyl cellulose to form a drypowdered blend;

(2) adding the dry powdered blend to water to form a slurry;

(3) adding water to the slurry with stirring to form a suspension; and

(4) applying the suspension to the tablets.

An additional illustration of the present invention is a solid dosageform containing an active ingredient ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide,or a pharmaceutically acceptable salt thereof, in particular themethanesulfonate salt, wherein the dosage form is prepared by theprocess.

The present invention further relates to a novel amorphous form of thecompoundN-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate. This amorphous form is produced directly as a resultof the instant process of tablet formulation.

The amorphous form ofN-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate exhibits a lack of crystallinity. The lack ofcrystallinity was confirmed by X-ray analysis wherein he X-raydiffraction pattern showed an amorphous halo.

The X-ray powder diffraction (XRPD) pattern was collected using aPhiulips APD3720 Automated Powder Diffraction instrument with copper Kαradiation. Measurements were made from 2° to 40° (2 theta) with thesample maintained at ambient room temperature.

In addition, examination of the amorphous form under microscopy showedno biorefringence.

The amorphous form may be prepared by evaporating a concentratedsolution ofN-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate in 25% aqueous ethanol (980 mg/ml) at 40° C. to give asolid.

Granulation is the process of adding a solvent, such as water orwater/ethanol, to a powder mixture until granules are formed. Thegranulation step may be varied from 2 to 35 minutes, preferably 3 to 10minutes, most preferably 4 to 8 minutes. Preferably, the granules aredried using a fluid bed dryer or tray dryer. Milling of the driedgranules is accomplished using a Quadro Comill or Fitz mill. Thelubrication step is the process of adding lubricant to the mixture. Thelubrication step may be varied from 30 seconds to 20 minutes, preferablyabout 1 minute.

The disclosed process may be used to prepare solid dosage forms,particularly tablets or granules, for medicinal administration.

The term "tablet," as used herein, is intended to encompass compressedpharmaceutical dosage formulations of all shapes and sizes, whethercoated or uncoated. Substances which may be used for coating includehydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC),titanium dioxide, talc, sweeteners and colorants.

The term "active ingredient," as used herein includes both the free baseN-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl)-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methyl-propanamide,as well as the pharmaceutically acceptable salts thereof, in particular,N-[1(R)-f(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate and crystal forms thereof. A preferred crystal form foruse in the present invention is that designated Form I.

Preferred diluents include: lactose, microcrystalline cellulose, calciumphosphate(s), mannitol, powdered cellulose, pregelatinized starch andother suitable diluents (see, e.g., Remington's Pharmaceutical Sciences,18th Edition, 1990, p. 1635). Microcrystalline cellulose and calciumphosphate dibasic, are particularly preferred. Specifically,microcrystalline cellulose NF, especially Avicel PH101, the trademarkedname for microcrystalline cellulose NF manufactured by FMC Corp. ispreferred.

Preferred binders include pregelatinized starch, hydroxypropylmethylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone (PVP) andother known binders (see, e.g., Remington's Pharmaceutical Sciences,18th Edition, 1990, pp. 1635-1636) and mixtures thereof. Mostpreferably, pregelatinized starch as employed as a binder. Specifically,starch pregelatinized NF 1500 manufactured by Colorcon Corporation ismost preferred.

The disintegrant may be one or more of several starches, clays,celluloses, algins, gums or crosslinked polymers known to those skilledin the art (See, e.g., Remington's Pharmaceutical Sciences, 18thEdition, 1990, p. 1637) and mixtures thereof. Preferably, one or more ofseveral modified starches or modified cellulose polymers, such asmicrocrystalline cellulose and croscarmellose sodium, are used.Croscarmellose sodium Type A, commercially available under the tradename "Ac-di-sol," is particularly preferred.

Preferred lubricants include magnesium stearate, zinc stearate, calciumstearate, stearic acid, surface active agents such as sodium laurylsulfate, magnesium lauryl sulfate, propylene glycol, sodium dodecanesulfonate, sodium oleate sulfonate and sodium laurate mixed withstearates and talc, sodium stearyl fumarate, hydrogenated vegetableoils, glyceryl palmitostearate, glyceryl behenate, sodium benzoate,mineral oil, talc and other known lubricants (see, e.g., Remington'sPharmaceutical Sciences, 18th Edition, 1990, pp. 1636-1637), andmixtures thereof. An especially preferred lubricant is magnesiumstearate.

The active ingredient,N-[1(R)-[(1,2-di-hydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl)-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide,may be prepared according to the methods disclosed in U.S. Pat. No.5,536,716, PCT Patent Publication WO 94/13696 and the methods disclosedherein.

The pharmaceutically acceptable salts ofN-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methylpropanamidemay be employed in the instant invention. Examples of pharmaceuticallyacceptable salts include the pharmaceutically acceptable acid additionsalts, such as the salts derived from using inorganic and organic acids.Examples of such acids are hydrochloric, nitric, sulfuric, phosphoric,formic, acetic, trifluoroacetic, propionic, maleic, succinic, malonic,methane sulfonic and the like.

The pharmaceutical compositions of the present invention comprise 0.1 to50% by weight of an active ingredient,N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide,or a pharmaceutically acceptable salt thereof, preferablyN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate; 0 to 77% by weight of a binder/diluent; 0 to 77% byweight of a first diluent; 0 to 77% by weight of a second diluent; 0 to6% by weight of a disintegrant; and 0 to 5% by weight of a lubricant. Itwill be appreciated by one skilled in the art that the sum of theproportions of the active ingredient, the binder/diluent, the firstdiluent, the second diluent, the disintegrant, and the lubricant are notgreater than 100% by weight.

More specifically, the binder/diluent is selected from hydroxy-propylmethylcellulose, hydroxypropyl cellulose, pregelatinized starch orpolyvinylpyrrolidone; the first and second diluents are independentlyselected from lactose, microcrystalline cellulose, calcium phosphatedibasic, mannitol, powdered cellulose or pregelatinized starch; thedisintegrant is selected from microcrystalline or croscarmellose sodium;and the lubricant is selected from magnesium stearate, calcium stearate,steric acid or a surface active agent.

In a specific embodiment, the binder/diluent is pregelatinized starch;the first diluent is microcrystalline cellulose; the second diluent iscalcium phosphate dibasic; the disintegrant is croscarmellose sodium;and the lubricant is magnesium stearate.

The pharmaceutical compositions of the present invention are preferablyin the form of tablets. The tablets may be, for example, from 50 mg to1.0 g in net weight, preferably 100 to 800 mg net weight, morepreferably 100 to 400 mg net weight.

Preferred pharmaceutical compositions comprise about 1 to 30% by weightof the active ingredient; about 20 to 40% by weight of pregelatinizedstarch; about 10 to 20% by weight of microcrystalline cellulose; about20 to 50% by weight of calcium phosphate dibasic; about 5 to 15% byweight of croscarmellose sodium; and about 0.05 to 5% by weight ofmagnesium stearate.

It will be appreciated by one skilled in the art that the sum of theabove proportions of the active ingredient, pregelatinized starch,microcrystalline cellulose, calcium phosphate dibasic, croscarmellosesodium, and magnesium stearate are not greater than 100% by weight.

More preferred pharmaceutical compositions in accordance with thepresent invention include those comprising the noted ingredients:

(1) about 1 to 2% by weight of the active ingredient; about 25 to 35% byweight of pregelatinized starch; about 10 to 20% by weight ofmicrocrystalline cellulose; about 45-55% by weight of calcium phosphatedibasic; about 4 to 8% by weight of croscarmellose sodium; and about 0.1to 1% by weight of magnesium stearate.

(2) about 5 to 10% by weight of the active ingredient, about 25 to 35%by weight of pregelatinized starch; about 10 to 20% by weight ofmicrocrystalline cellulose; about 40 to 50% by weight of calciumphosphate dibasic; about 4 to 8% by weight of croscarmellose sodium; andabout 0.1 to 1% by weight of magnesium stearate.

(3) about 25 to 35% by weight of the active ingredient, about 15 to 25%by weight of pregelatinized starch; about 10 to 20% by weight ofmicrocrystalline cellulose; about 15-25% by weight of calcium phosphatedibasic; about 10 to 20% by weight of croscarmellose sodium; and about0.1 to 1% by weight of magnesium stearate.

It will be appreciated by one skilled in the art that the sum of theabove proportions of the active ingredient, pregelatinized starch,microcrystalline cellulose, calcium phosphate dibasic, croscarmellosesodium, and magnesium stearate are not greater than 100% by weight.

Especially referred pharmaceutical compositions as envisioned forcommercial development are as follows:

Tablets of 1.0 mg potency free base:

about 1.18% by weight of active ingredient as the methanesulfonate salt;about 30.0% by weight of pregelatinized starch; about 15.0% by weight ofmicrocrystalline cellulose; about 47.3% by weight of calcium phosphatedibasic; about 6.0% by weight of croscarmellose sodium; and about 0.5%by weight of magnesium stearate. This composition comprises about 1.2 mgof active ingredient as the methanesulfonate salt; about 30 mg ofpregelatinized starch; about 15 mg of microcrystalline cellulose; about47.3 mg of calcium phosphate dibasic; about 6.0 mg of croscarmellosesodium; and about 0.5 mg of magnesium stearate per dosage unit.

Optionally, the 1.0 mg potency tablet may be coated with a coatingcomprising about 0.8% by weight of hydroxypropyl methylcellulose; about0.8% by weight of hydroxypropyl cellulose; about 0.32% by weight oftitanium dioxide; and about 0.08% by weight of talc (as a percentage ofthe core tablet weight).

Tablets of 5.0 mg potency free base:

about 1.48% by weight of active ingredient as the methanesulfonate salt;about 30.0% by weight of pregelatinized starch; about 15.0% by weight ofmicrocrystalline cellulose; about 47.0% by weight of calcium phosphatedibasic; about 6.0% by weight of croscarmellose sodium; and about 0.5%by weight of magnesium stearate. This composition comprises about 6 mgof active ingredient as the methanesulfonate salt; about 120 mg ofpregelatinized starch; about 60 mg of microcrystalline cellulose; about188 mg of calcium phosphate dibasic; about 24 mg of croscarmellosesodium; and about 2 mg of magnesium stearate per dosage unit.

Optionally, the 5.0 mg potency tablet may be coated with a coatingcomprising about 0.8% by weight of hydroxypropyl methylcellulose; about0.8% by weight of hydroxypropyl cellulose; about 0.32% by weight oftitanium dioxide; and about 0.08% by weight of talc (as a percentage ofthe core tablet weight).

Tablets of 25 mg potency free base:

about 7.39% by weight of active ingredient as the methanesulfonate salt;about 282% by weight of pregelatinized starch; about 14.2% by weight ofmicrocrystalline cellulose; about 43.6% by weight of calcium phosphatedibasic; about 6.0% by weight of croscarmellose sodium; and about 0.5%by weight of magnesium stearate. This composition comprises about 30 mgof active ingredient as the methanesulfonate salt; about 113 mg ofpregelatinized starch; about 57 mg of microcrystalline cellulose; about174 mg of calcium phosphate dibasic; about 24 mg of croscarmellosesodium; and about 2 mg of magnesium stearate per dosage unit.

Optionally, the 25 mg potency tablet may be coated with a coatingcomprising about 0.8% by weight of hydroxypropyl methylcellulose; about0.8% by weight of hydroxypropyl cellulose; about 0.32% by weight oftitanium dioxide; and about 0.08% by weight of talc (as a percentage ofthe core tablet weight).

Tablets of 100 mg potency free base:

about 29.5% by weight of active ingredient as the methanesulfonate salt;about 19.5% by weight of pregelatinized starch; about 15.0% by weight ofmicrocrystalline cellulose; about 20.4% by weight of calcium phosphatedibasic; about 15.0% by weight of croscarmellose sodium; and about 0.5%oby weight of magnesium stearate. This composition comprises about 118 mgof active ingredient as the methanesulfonate salt; about 78 mg ofpregelatinized starch; about 60 mg of microcrystalline cellulose; about82 mg of calcium phosphate dibasic; about 60 mg of croscarmellosesodium; and about 2 mg of magnesium stearate per dosage unit.

Optionally, the 100 mg potency tablet may be coated with a coatingcomprising about 0.8% by weight of hydroxypropyl methylcellulose; about0.8% by weight of hydroxypropyl cellulose; about 0.32% by weight oftitanium dioxide; and about 0.08% by weight of talc (as a percentage ofthe core tablet weight).

The tablets of the 1.0 mg potency are preferably formulated in an 100 mgtablet by using 30 μl of a solution of 25% ethanol/75% water per tablet.The tablets of the 5.0 mg potency are preferably formulated in an 400 mgtablet by using 120 μl of a solution of 25% ethanol/75% water pertablet. The tablets of the 25 mg potency are preferably formulated in an400 mg tablet by using 120 μl of a solution of 25% ethanol/75% water pertablet. The tablets of the 100 mg potency are preferably formulated inan 400 mg tablet by using 120 μl of a solution of 25% ethanol/75% waterper tablet.

In a particularly preferred embodiment, the tablet formulations of theinstant invention are coated. In the pharmaceutical compositionsenvisioned for commercial development described above, the tablets of1.0 mg, 5.0 mg, 25 mg and 100 mg potency free base are coated with about0.8% by weight of hydroxypropyl methylcellulose; about 0.8% by weight ofhydroxypropyl cellulose; about 0.32% by weight titanium dioxide; andabout 0.08% by weight of purified talc.

In the most preferred embodiment, the active ingredient in theabove-described pharmaceutical compositions isN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl)-2-amino-2-methylpropanamidemethanesulfonate.

The compositions of the present invention are in a form for oraladministration and may take the form of tablets, capsules, granules,powders, tablets or granules for buccal administration, or liquidpreparations such as suspensions. Granules and powders may be ingesteddirectly, or dispersed in water or other suitable vehicle prior toadministration. Capsules may be of the hard or soft gelatin type,including soft gelatin capsules.

The pharmaceutical compositions of the present invention may alsocontain other excepients conventional in the art such as flavorings,sweeteners, and the like. Suitable flavorings include for example fruitflavors or natural or synthetic mint or peppermint flavors. Suitablesweeteners include for example sugar, saccharin or aspartame.

The utility of the active ingredient of the formulation of the presentinvention as growth hormone secretagogues may be demonstrated bymethodology known in the art, such as an assay described by Smith, etal., Science, 260, 1640-1643 (1993) (see text of FIG. 2 therein). Inparticular, the active ingredient used in the formulation the presentinvention had activity as a growth hormone secretagogue in theaforementioned assay. Such a result is indicative of the activity of theformulation of the present invention as a growth hormone secretagogue.

The formulations of the present invention may be administered toanimals, including man, to release growth hormone in vivo. For example,the formulations can be administered to commercially important animalssuch as swine, cattle, sheep and the like to accelerate and increasetheir rate and extent of growth, to improve feed efficiency and toincrease milk production in such animals. In addition, theseformulations can be administered to humans in vivo as a diagnostic toolto directly determine whether the pituitary is capable of releasinggrowth hormone. For example, the formulation of the present inventioncan be administered in vivo to children. Serum samples taken before andafter such administration can be assayed for growth hormone. Comparisonof the amounts of growth hormone in each of these samples would be ameans for directly determining the ability of the patient's pituitary torelease growth hormone.

Accordingly, the present invention includes within its scopepharmaceutical compositions comprising, as an active ingredient, thecompoundN-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methyl-propanamidein association with a pharmaceutical carrier or diluent. Optionally, theactive ingredient of the pharmaceutical compositions may comprise ananabolic agent in addition to the compoundN-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenyl-methyl-oxy)ethyl]-2-amino-2-methyl-propanamideor another composition which exhibits a different activity, e.g., anantibiotic growth permittant or an agent to treat osteoporosis or incombination with a corticosteroid to minimize the catabolic side effectsor with other pharmaceutically active materials wherein the combinationenhances efficacy and minimizes side effects.

Growth promoting and anabolic agents include, but are not limited toTRH, diethylstilbesterol, amino acids, estrogens, β-agonists,theophylline, anabolic steroids, enkephalins, E series prostaglandins,compounds disclosed in U.S. Pat. No. 3,239,345, e.g., zeranol, andcompounds disclosed in U.S. Pat. No. 4,036,979, e.g., sulbenox orpeptides disclosed in U.S. Pat. No. 4,411,890.

A still further use of the formulations of this invention is incombination with other growth hormone secretagogues such as the growthhormone releasing peptides GHRP-6, GHRP-1 as described in U.S. Pat. No.4,411,890 and publications WO 89/07110, WO 89/07111 and B-HT920 as wellas hexarelin and GHRP-2 as described in WO 93/04081 or growth hormonereleasing hormone (GHRH, also designated GRF) and its analogs or growthhormone and its analogs or somatomedins including IGF-1 and IGF-2 orα-adrenergic agonists such as clonidine or serotonin 5HTID agonists suchas sumitriptan or agents which inhibit somatostatin or its release suchas physostigmine and pyridostigmine. In particular, the formulations ofthis invention may be used in combination with growth hormone releasingfactor, an analog of growth hormone releasing factor, IGF-1, or IGF-2.For example, a formulation of the present invention may be used incombination with IGF-1 for the treatment or prevention of obesity. Inaddition, a formulation of this invention may be employed in conjunctionwith retinoic acid to improve the condition of musculature and skin thatresults from intrinsic aging.

As is well known to those skilled in the art, the known and potentialuses of growth hormone are varied and multitudinous. The administrationof the formulations of this invention for purposes of stimulating therelease of endogenous growth hormone can have the same effects or usesas growth hormone itself. These varied uses of the present formulationsthus may be summarized as follows: stimulating growth hormone release inelderly humans; treating growth hormone deficient adults; prevention ofcatabolic side effects of glucocorticoids; treatment of osteoporosis;stimulation of the immune system, acceleration of wound healing;accelerating bone fracture repair; treatment of growth retardation;treating acute or chronic renal failure or insufficiency; treatment ofphysiological short stature, including growth hormone deficientchildren; treating short stature associated with chronic illness;treatment of obesity and growth retardation associated with obesity;treating growth retardation associated with Prader-Willi syndrome andTurner's syndrome; accelerating the recovery and reducinghospitalization of burn patients or following major surgery such asgastrointestinal surgery; treatment of intrauterine growth retardation,and skeletal dysplasia, treatment of peripheral neuropathies;replacement of growth hormone in stressed patients; treatment ofosteochondrody-splasias, Noonans syndrome, schizophrenia, depression,Alzheimer's disease, delayed wound healing, and psychosocialdeprivation; treatment of pulmonary dysfunction and ventilatordependency; attenuation of protein catabolic response after a majoroperation; treating malabsorption syndromes; reducing cachexia andprotein loss due to chronic illness such as cancer or AIDS; acceleratingweight gain and protein accretion in patients on TPN (total parenteralnutrition); treatment of hyperinsulinemia including nesidioblastosis;adjuvant treatment for ovulation induction and to prevent and treatgastric and duodenal ulcers; to stimulate thymic development and preventthe age-related decline of thymic function; adjunctive therapy forpatients on chronic hemodialysis; treatment of immunosuppressed patientsand to enhance antibody response following vaccination; increasing thetotal lymphocyte count of a human, in particular, increasing the T₄ /T₈-cell ratio in a human with a depressed T₄ /T₈ -cell ratio resulting,for example, from physical trauma, such as closed head injury, or frominfection, such as bacterial or viral infection, especially infectionwith the human immunodeficiency virus; improvement in muscle strength,mobility, maintenance of skin thickness, metabolic homeostasis, renalhemeostasis in the frail elderly; stimulation of osteoblasts, boneremodelling, and cartilage growth; stimulation of the immune system incompanion animals and treatment of disorders of aging in companionanimals; growth promotant in livestock; and stimulation of wool growthin sheep. Further, the instant compounds are useful for increasing feedefficiency, promoting growth, increasing milk production and improvingthe carcass quality of livestock. Likewise, the instant formulations areuseful in a method of treatment of diseases or conditions which arebenefited by the anabolic effects of enhanced growth hormone levels.

In particular, the instant formulations are useful in the prevention ortreatment of a condition selected from the group consisting of:osteoporosis; catabolic illness; immune deficiency, including that inindividuals with a depressed T₄ /T₈ cell ratio; hip fracture;musculoskeletal impairment in the elderly; growth hormone deficiency inadults or in children; obesity; cachexia and protein loss due to chronicillness such as AIDS or cancer; and treating patients recovering frommajor surgery, wounds or burns, in a patient in need thereof.

In addition, the instant formulations may be useful in the treatment ofillnesses induced or facilitated by corticotropin releasing factor orstress- and anxiety-related disorders, including stress-induceddepression and headache, abdominal bowel syndrome, immune suppression,HIV infections, Alzheimer's disease, gastrointestinal disease, anorexianervosa, hemorrhagic stress, drug and alcohol withdrawal symptoms, drugaddiction, and fertility problems.

It will be known to those skilled in the art that there are numerouscompounds now being used in an effort to treat the diseases ortherapeutic indications enumerated above. Combinations of thesetherapeutic agents some of which have also been mentioned above in andwith the formulations of this invention will bring additional,complementary, and often synergistic properties to enhance the growthpromotant, anabolic and desirable properties of these varioustherapeutic agents. In these combinations, the therapeutic agents andthe active ingredient in the formulations of this invention may beindependently present in dose ranges from one one-hundredth to one timesthe dose levels which are effective when these compounds and activeingredients are used singly.

Combined therapy to inhibit bone resorption, prevent osteoporosis andenhance the healing of bone fractures can be illustrated by combinationsof bisphosphonates and the formulations of this invention. The use ofbisphosphonates for these utilities has been reviewed, for example, byHamdy, N. A. T., Role of Bisphosphonates in Metabolic Bone Diseases,Trends in Endocrinol. Metab., 4, 19-25 (1993). Bisphosphonates withthese utilities include alendronate, tiludronate, dimethyl-APD,risedronate, etidronate, YM-175, clodronate, pamidronate, and BM-210995.According to their potency, oral daily dosage levels of thebisphosphonate of between 0.1 mg and 5 g and daily dosage levels of theactive ingredient of the formulation of this invention of between 0.01mg/kg to 20 mg/kg of body weight are administered to patients to obtaineffective treatment of osteoporosis.

In the case of alendronate daily oral dosage levels of 0.1 mg to 50 mgare combined for effective osteoporosis therapy with 0.01 mg/kg to 20mg/kg of the active ingredient employed in the formulation of thisinvention. Osteoporosis and other bone disorders may also be treatedwith the formulations of this invention in combination with calcitonin,estrogens, raloxifene and calcium supplements such as calcium citrate.

Anabolic effects especially in the treatment of geriatric male patientsare obtained with formulations of this invention in combination withanabolic steroids such as oxymetholone, methyltesterone, fluoxymesteroneand stanozolol.

The pharmaceutical tablet compositions of the present invention may alsocontain one or more additional formulation ingredients selected from awide variety of excipients (also referred to as "additives") known inthe pharmaceutical formulation art. According to the desired propertiesof the tablet, any number of additives may be selected, alone or incombination, based upon their known uses in preparing tabletcompositions. Such additives include, but are not limited to, diluents,binders, compression aids, disintegrants, lubricants, flavors, flavorenhancers, sweeteners and preservatives. Due to the bitter taste of theactive ingredient, the inclusion of a sweetener may be desired.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient be such that a suitable dosage form is obtained. The selecteddosage depends upon the desired therapeutic effect, on the route -ofadministration, and on the duration of the treatment. Generally, dosagelevels of between 0.0001 to 10 mg/kg. of body weight daily areadministered to patients and animals, e.g., mammals, to obtain effectiverelease of growth hormone. Preferably, the dosage level will be about0.001 to about 25 mg/kg per day; more preferably about 0.01 to about 10mg/kg per day.

Methods for preparing the formulations of the present invention, as wellas the active ingredient are illustrated in the following Examples. Thefollowing examples are given for the purpose of illustrating the presentinvention and shall not be construed as being limitations on the scopeor spirit of the instant invention.

EXAMPLE 1

N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide

Step A:1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdine]hydrochloride

To a solution of 1.20 g (5.8 mmol) of1'-methyl-1,2-dihydro-spiro[3H-indole-3,4'-piperdine] (prepared asdescribed by H. Ong, et al., J. Med. Chem., 23, 981-986 (1983)) in 20 mLof dry dichloromethane at 0° C. was added triethylamine (0.90 mL; 6.4mmol) and methanesulfonyl chloride (0.49 mL; 6.35 mmol) and stirred for30 min. The reaction mixture was poured into 15 mL of saturated aqueoussodium bicarbonate solution and extracted with dichloromethane (2×10mL). The combined organics were washed with brine (20 mL), dried overanhydrous potassium carbonate, filtered and the solvent removed underreduced pressure to yield 1.44 g of the methanesulfonamide derivative aspale yellow oil which was used without purification.

To a solution of above crude product in 20 mL of dry 1,2-dichloroethaneat 0° C. was added 1.0 mL (9.30 mmol) of 1-chloroethyl chloroformate,and then stirred at RT for 30 min and finally at reflux for 1 h. Thereaction mixture was concentrated to approximately one third of thevolume and then diluted with 20 mL of dry methanol and refluxed for 1.5h. The reaction was cooled to RT and concentrated to approximately onehalf of the volume. The precipitate was filtered and washed with a smallvolume of cold methanol. This yielded 1.0 g of the piperidine HCl saltas a white solid. The filtrate was concentrated and a small volume ofmethanol was added followed by ether. The precipitated material was onceagain filtered, washed with cold methanol, and dried. This gave anadditional 0.49 g of the desired product. Total yield 1.49 g (70%).

¹ H NMR (CDCl₃, 200 MHz) δ 7.43-7.20 (m, 3H), 7.10 (dd, 1H), 3.98 (bs,2H), 3.55-3.40 (bd, 2H), 3.35-3.10 (m, 2H), 2.99 (s, 3H), 2.15 (t, 2H),2.00 (t, 2H).

Step B:N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-[(1,1-dimethylethoxy)carbonyl]amino-2-methyl-propanamide

To 0.35 g (1.15 mmol) of(2R)-2-[(1,1-dimethylethoxy)carbonyl]amino-3-[2-(phenylmethyloxy)ethyl]-1-propanoicacid in 13 mL of dichloromethane was added1,2-dihydro-1-methanesulfonylspiro-[3H-indole-3,4'-piperdine)hydrochloride (0.325 g; 1.07 mmol), 0.18 -mL (1.63 mmol) ofN-methylmorpholine, 0.159 g (1.18 mmol) of 1-hydroxybenztriazole(HOBT)and stirred for 15 min. EDC (0.31 g; 1.62 mol) was added and stirringwas continued for 1 h. An additional 60 μL of N-methylmorpholine wasadded and stirred for 45 min. The reaction mixture was poured into 5 mLof water and the organic layer was separated. The organic layer waswashed with 5 mL of 0.5N aqueous hydrochloric acid and 5 mL of saturatedaqueous sodium bicarbonate solution. The combined organics were driedover anhydrous magnesium sulfate, and concentrated to yield 0.627 g ofthe product as a yellow foam which was used without purification.

To a 0.627 g (1.07 mmol) of the above product in 5 mL of dichloromethanewas added 1.0 mL of trifluoroacetic acid and stirred at RT for 75 min.An additional 1.00 mL of trifluoroacetic acid was added and stirred for10 min. The reaction mixture was concentrated, diluted with 5.0 mL ofdichloromethane and carefully basified by pouring into 10 mL of 10%aqueous sodium carbonate solution. The organic layer was separated andthe aqueous layer was further extracted with 2×15 mL of dichloromethane.The combined organics were washed with 5 mL of water, dried overpotassium carbonate, filtered and concentrated to give the 0.486 g ofthe amine as a light yellow foam which was used without purification.

To 0.486 g (1.01 mmol) of the amine and 10 mL of dichloromethane wasadded 0.26 g (1.28 mmol) of2-[(1,1-dimethylethoxy)carbonyl]amino-2-methyl-propanoic acid, 0.173 g(1.28 mmol) of 1-hydroxybenztriazole (HOBT) and EDC (0.245 g; 1.28 mol)and stirried at RT overnight. The reaction mixture was poured into 5.0mL of water and the organic layer was separated. The aqueous layer wasback extracted with 5 mL of dichloromethane. The combined organics werewashed with 5.0 mL of 0.5N aqueous hydrochloric acid, 5 mL of saturatedaqueous sodium bicarbonate solution dried over anhydrous magnesiumsulfate, and concentrated to yield 0.751 g of the crude product as ayellow foam. A solution of this crude product in dichloromethane waschromatographed on 25 g of silica gel and eluted first withhexanes/acetone/dichloromethane (70/25/5) and then withhexanes/acetone/dichloromethane (65/30/5). This gave 0.63 g of the titlecompound as a white solid.

¹ H NMR (CDCl₃, 400 MHz) Compound exists as a 3:2 mixture of rotamers δ7.40-7.10 (m, 6H), 7.06 (d, 1/3H), 7.02 (t, 1/3H), 6.90 (t, 1/3H), 6.55(d, 1/3H), 5.15 (m, 1H), 4.95 (bs, 1H), 4.63 (bd, 1/3H), 4.574.40 (m, 22/3 H), 4.10 (bd, 1/3H), 4.00 (bd, 1/3H), 3.82 (t, 1H), 3.78-3.62 (m,2H), 3.60-3.50 (m, 1H), 3.04 (q, 1H), 2.87 (s, 1H), 2.86 (s, 2H),2.80-2.60 (m, 1H), 1.90 (bs, 1H), 2.85-2.75 (m, 1H), 1.82-1.60 (m, 3H),1.55-1.45 (m, 1H), 1.45 (s, 4H), 1.42 (s, 2H), 1.39 (s, 9H).

Step C:N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperidin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidehydrochloride

To 0.637 g (0.101 mmol) of the intermediate from Step B in 5 mL ofdichloromethane was added 2.5 mL of trifluoroacetic acid and stirred atRT for 30 min. The reaction mixture was concentrated to an oil, taken upin 10 mL of ethyl acetate and washed with 8 mL of 10% aqueous sodiumcarbonate solution. The aqueous layer was further extracted with 5 mL ofethyl acetate. The combined organics were washed with 10 mL of water,dried over magnesium sulfate, filtered and concentrated to give the0.512 g of the free base as a white foam.

To 0.512 g of the free base in 5 mL of ethyl acetate at 0° C. was added0.2 mL of saturated hydrochloric acid in ethyl acetate and stirred for1.5 h. The white precipitate was filtered under nitrogen, washed withether, and dried to give 0.50 g of the title compound as a white solid.

¹ H NMR (400 MHz, CD₃ OD) Compound exists as 3:2 mixture of rotamers. δ7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7.08 (t, 1/3H), 7.00 (t, 1/3H),6.80 (d, 1/3H), 5.16 (ddd, 1H), 4.60-4.42 (m, 3H), 4.05 (t, 1H), 3.90(bs, 2H), 3.83-3.70 (m, 2H), 3.30-3.15 (m, 1H), 2.97 (s, 1H), 2.95 (s,2H), 2.90-2.78 (m, 1H), 1.96 (t, 1/3H), 1.85-1.65 (m, 4H), 1.63 (s, 2H),1.60 (s, 4H).

EXAMPLE 2

N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide

Step A:(2R)-[[[-2-(1,1-dimethylethoxy)carbonyl]amino]-2,2-dimethyl-1-oxoethyl]amino-2-(phenylmethoxy)ethyl]-1-propanoicacid allyl ester

Prepared from(2R)-2-[(1,1-dimethylethoxy)carbonyl]-amino-3-(phenylmethyloxy)ethyl-propanoicacid and allyl alcohol by carrying out the coupling reaction in CH₂ Cl₂in the presence of EDC and DMAP.

¹ H NMR (400 MHz, CDCl₃) δ 7.25 (s, 5H), 5.8 (m, 1H), 5.2 (dd, 2H), 5.0(bs, 1H), 4.7 (m, 1H), 4.6 (m, 2H), 4.4 (dd, 2H), 3.9 (dd, 1H), 3.6 (dd,1H), 1.45 (d, 6H), 1.39 (s, 9H).

Step B:(2R)-[[[-2-(1,1-dimethylethoxy)carbonyl]amino]-2,2-dimethyl-1-oxoethyl]amino-2-(phenylmethyloxy)ethyl)-1-propanoic acid

To a stirred solution of the crude intermediate obtained in Step A (6.7g, 15.9 mmol), tetrakis (triphenylphosphine)-palladium (1.8 g, 0.1 eq)and, triphenyl phosphine (1.25 g, 0.3 eq) was added a solution ofpotassium-2-ethyl hexanoate (35 mL, 0.5M solution in EtOAc). Thereaction mixture was stirred at room temperature under nitrogenatmosphere for 1 h and then diluted with ether (100 mL) and poured intoice-water. The organic layer was seperated and the aqueous fraction wasacidified with citric acid (20%), then extracted with EtOAc. The EtOAcextracts were washed with brine, dried over magnesium sulfate, filteredand evaporated to give the title compound as a solid.

¹ H NMR (400Hz, CD₃ OD) δ 7.3 (s, 5H), 4.7 (m, 1H), 4.5 (s, 2H), 4.0 (m,1H), 3.6 (m, 1H), 1.4 (d, 6H), 1.3 (s, 9H).

Step C:N-[(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-[(1,1-dimethyl-ethoxy)carbonyl]amino-2-methyl-propanamide

To a solution of 1.0 g (3.44 mmol) of1-methanesulfonylspiro[indoline-3,4'-piperidine] hydrochloride, 1.44 g(3.78 mmol) of(2R)-[[-2-(1,1-dimethylethoxy)carbonyl)amino]-2,2-dimethyl-1-oxo-ethyl]-amino-2-(phenylmethyloxy)ethyl)-1-propanoicacid, N-methyl morpholine (0.58 mL; 5.20 mmol), and1-hydroxybenztriazole (HOBT) (0.58 g; 3.78 mmol), in 50 mL ofdichloromethane was added EDC (1.03 g; 5.20 mmol) and stirred at RT for16 h. The reaction mixture was diluted with an additional 50 mL ofdichloromethane and washed with aqueous sodium bicarbonate solution (50mL), dried over anhydrous magnesium sulfate, filtered, and concentrated.Flash chromatography (50 g silica gel) of the crude oily residue gave2.148 g (90%) of the desired material as a colorless foam.

¹ H NMR (CDCl₃, 400 MHz) Compound exists as a 3:2 mixture of rotamers δ7.40-7.10 (m, 6H), 7.06 (d, 1/3H), 7.02 (t, 1/3H), 6.90 (t, 1/3H), 6.55(d, 1/3H), 5.15 (m, 1H), 4.95 (bs, 1H), 4.63 (bd, 1/3H), 4.574.40 (m, 22/3 H), 4.10 (bd, 1/3H), 4.00 (bd, 1/3H), 3.82 (t, 1H), 3.78-3.62 (m,2H), 3.60-3.50 (m, 1H), 3.04 (q, 1H), 2.87 (s, 1H), 2.86 (s, 2H),2.80-2.60 (m, 1H), 1.90 (bs, 1H), 2.85-2.75 (m, 1H), 1.82-1.60 (m, 3H),1.55-1.45 (m, 1H), 1.45 (s, 4H), 1.42 (s, 2H), 1.39 (s, 9H).

Step D:N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidehydrochloride

To a solution of 2.148 g (3.41 mmol) of the intermediate from Step C in10 mL of dichloromethane was added 5 mL of trifluoroacetic acid andstirred for 1 h. The reaction mixture was concentrated and basified with100 mL of 5% aqueous sodium carbonate solution and extracted withdichloromethane (3×50 mL). The combined organics were washed with brine(50 mL), dried over anhydrous potassium carbonate, filtered, andconcentrated to yield a colorless foam. To a solution of the foam in 25mL of ethyl acetate at 0° C. was added 4 mL of 1 M solution ofhydrochloric acid in ethyl acetate. The precipitate was filtered andwashed first with ethyl acetate and then with ethyl acetate-ether (1:1),dried to yield 1.79 g (93%) of the title compound as a colorless solid.

¹ H NMR (400 MHz, CD₃ OD) Compound exists as 3:2 mixture of rotamers. δ7.40-7.28 (m, 4H), 7.25-7.17 (m, 2H), 7.08 (t, 1/3H), 7) (t, 1/3H), 6.80(d, 1/3H), 5.16 (ddd, 1H), 4.604.42 (m, 3H), 4.05 (1H), 3.90 (bs, 2H),3.83-3.70 (m, 2H), 3.30-3.15 (m, 1H0, 2.97 (s, 1H), 2.95 (s, 2H),2.90-2.78 (m, 1H), 1.96 (t, 1/3H), 1.85-1.65 (m, 4H), 1.63 (s, 2H), 1.60(s, 4H).

EXAMPLE 3

N-[1(R)-[(1,2-Dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemesylate

This compound was prepared by the treating the free base obtained inExample 5, Step D, with methane sulfonic acid. The title compound wasobtained by recrystallizing it from ethyl acetate-ethanol-water.m.p.=166°-168° C.

EXAMPLE 4 ##STR2## Isonipecotic acid-N-benzyl carbamate (3)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Isonipecotic acid (2) T.C.I.                                                                         4.02 kg (31.1 mol)                                     Benzyl chloroformate (Schweitzerhall)                                                                6.91 kg (40.5 mol)                                     K.sub.2 CO.sub.3       10.1 kg (72.9 mol)                                     Water                  40.2 L                                                 ______________________________________                                    

Isonipecotic acid (2) and K₂ CO₃ were dissolved in 40.2 L of water in a100 L 4 neck flask with mechanical stirring under N₂ and the solutionwas cooled to 10° C. Benzyl chlorofornate was added, maintaining thetemperature between 9 and 14° C., and the mixture was warmed up to 22°C. after the addition was complete and aged for 58 h. The addition wascompleted in 4 h at which point the pH was 9.0. After aging for 58 hthere was no change in the pH.

The reaction mixture was transferred to a 200 L extractor and washedwith 3×13 kg (15 L) of IPAC and 1×12 L of EtOAc. The aqueous layer wasextracted with 8 L of toluene. After the washes the benzyl alcoholcontent was reduced from 3.8% to 1.4% by HPLC analysis. HPLC analytical:Dupont Zorbax 25 cm RXC8 column with 1.5 ml/min flow and detection at254 nm; isocratic mixture with 35% MeCN, 65% of 0.1% aqueous H₃ PO₄ ;retention times: 3=6.9 min, benzyl alcohol=3.3 min, toluene=17.3 min.

The aqueous phase was acidified with 37% aqueous HCl to pH 1.8. Carbondioxide was evolved during the addition of HCl, but gas evolution waseasily controlled. The addition of HCl took <1 h and required 10 L ofconc. HCl. The aqueous phase was extracted with 3×6.6 L of toluene. Thetoluene extracts were dried with 2 kg of sodium sulfate and filteredthrough a pad of Solka-floc™. The combined filtrates weighed 17.8 kg.The crude yield of carbamate 3 was 7.89 kg (97%) (as obtained byevaporation of weighed aliquots of the filtrates to dryness). Thefiltrates were transferred through a 10μ inline filter to a 100 L flask.The extracts were concentrated at 10 mbar at <25° C. to a volume of 18L. The final concentration of carbamate 3 was 440 g/L. The concentrationof the toluene filtrate served to azeotropically remove final traces ofwater (final KF=170 mg/L). The product was 99.1 area % pure with 0.9area % benzyl alcohol as the only impurity.

EXAMPLE 5 ##STR3##

Isonipecotic acid chloride-N-benzyl carbamate (4)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Isonipecotic acid N-benzyl carbamate                                                                 7.89 kg (30.0 mol) in                                  in toluene. (MW = 263.30)                                                                            17.9 L                                                 Oxalyl chloride (MW = 126.93)                                                                        3.94 kg (31.0 mol)                                     DMF (MW = 73.10)       10 mL                                                  Toluene                12 L                                                   ______________________________________                                    

To the toluene solution of benzyl carbamate 3 from the preceding stepwas added 5 mL of DMF and 10 L of toluene. The oxalyl chloride was addedover a period of 20 min. The reaction mixture was aged for 16 h at 18°C. under a slow stream of nitrogen. HPLC analysis of the reactionmixture showed that 1.3% of the carboxylic acid 3 still remainedunreacted. The reaction mixture was warmed to 26° C., and 5 mL of DMFwere added. The mixture was aged for 2.5 h. A 1.0 mL aliquot of thereaction mixture was quenched with 5.0 mL of tert-butylamine andanalyzed after evaporation by HPLC: 25 cm Dupont Zorbax RXC8 column at50° C. with 1 mL/min flow and detection at 220 nm; isocratic 42% MeCN,58% of 0.1% aqueous H₃ PO₄. This method showed that <0.05% of the acid 3remained (as judged by A) and showed >3 area % B (>1 mol % (COCl)₂).##STR4##

The mixture was concentrated at 10 mbar and a temperature of 20-25° C.until 5 L of solvent had been removed.

The typical HPLC profile of concentrated toluene solution after t-BuNH₂quench described above is as follows:

    ______________________________________                                        Retention time (min)                                                                         Area %   Identity                                              ______________________________________                                        2.1            <0.5%    carboxylic acid 3                                     7.8            <0.5%    benzyl chloride                                       11.0           >99%     Cbz-t-butylcarboxamide A                              12.1           NA       toluene                                               12.7           <0.5%    ditert-butyloxamide B                                 ______________________________________                                    

EXAMPLE 6 ##STR5## Piperidine-4-carboxaldehyde-1-benzyl carbamate (5)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Isonipecotic acid chloride N-benzyl carbamate (4)                                                     3.38 kg (12.0                                         mol)                                                                          in toluene (MW = 281.74)                                                                              in 5.54 kg                                            DIEA (KF = 18 mg/L)     1.55 kg (15.0 mol)                                    10% Pd/C (KF <20 mg/g)  101 g                                                 thioanisole (MW = 124.21, d = 1.058)                                                                  0.56 g                                                ______________________________________                                    

The DIEA and thioanisole were added to the solution of (4) in toluenefrom the previous step and the catalyst was suspended in this mixture.The mixture was immediately placed into the 5 gal autoclave andhydrogenated at 20° C. and 40 psi of H₂. After 18 h the reaction hadtaken up 70% the theoretical amount of hydrogen and HPLC analysis of analiquot that was quenched with tert-butylamine indicated that 14.2 area% of acid chloride 2 remained. HPLC conditions same as above. Retentiontime: 5=8.1 min.

A second charge of catalyst (101 g) and thioanisole (0.54 g) were addedas a slurry in 1375 mL toluene to the hydrogenator. After 23 h HPLCanalysis of an aliquot that was quenched with tert-butylamine indicatedthat 1.8 area % of acid chloride 2 remained. The mixture was purged withnitrogen and the catalyst and precipitated DIEA·HCl were removed byfiltration through Solka-floc™. The filter cake was washed with 10 L oftoluene. The filtrates were transferred through a 10μ inline filter to a50 L extractor and washed with 2×7.2 L of 1 M aqueous HCl and 2×7.2 L ofwater. The mixture was concentrated at 10 mbar and a temperature of25-30° C. until 5 L of residue remained.

    ______________________________________                                        Retention time (min)                                                                           Area %    Identity                                           ______________________________________                                        2.1              <2        carboxylic acid 3                                  6.6              <1        dimer 21                                           8.1              >95       aldehyde 5                                         ______________________________________                                    

The assay yield of aldehyde 3 was 94% by HPLC analysis.

EXAMPLE 7 ##STR6## CBZ-Spiroindoline (9)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Piperidine-4-carboxaldehyde-1-benzyl                                                              1.71 kg (6.89 mol)                                        carbamate (5) in toluene solution                                                                 in 21.4 kg                                                Phenylhydrazine     900 mL, 981 g (9.15 mol)                                  Trifluoroacetic acid (TFA)                                                                        2.20 L, 3.26 kg (28.6 mol)                                NaBH.sub.4          300 g, (7.93 mol)                                         Toluene             34.4 kg                                                   MeCN                7.0 L                                                     MeOH                7.0 L                                                     ______________________________________                                    

The crude aldehyde 5 solution from the previous step was transferredthrough a 10μ inline filter to a 100 L reactor equipped with Tefloncoated copper coils for cooling or heating and a mechanical stirrer.Toluene (34.4 kg) and MeCN (7 L) were added, and the resulting solutionwas cooled to 0° C. Phenylhydrazine was added in portions and thetemperature was maintained at -1 to 3° C. while nitrogen wascontinuously bubbled through the reaction mixture.

The phenylhydrazine was added until TLC and HPLC analysis indicatedcomplete consumption of the aldehyde 5 and the appearance of a slightexcess (<5%) of phenylhydrazine. TLC conditions: Silica, E. MerckKieselgel G60 F254 0.25 mm; diethyl ether/pentane (4/1); and developingagent 0.5% ceric sulfate, 14% ammonium molybdate in 10% aqueous sulfuricacid then heat; Rf: aldehyde 5=0.52, phenylhydrazone 7=0.61,phenylhydrazine 6=0.21.

HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 30° C. with 1.0mL/min flow and detection at 254 nm; gradient schedule:

    ______________________________________                                        Time (min)    acetonitrile:water                                              ______________________________________                                        0             57:43                                                           10            65:35                                                           15            75:25                                                           18            75:25                                                           ______________________________________                                    

retention times: phenylhydrazine 6=4.5 min, toluene=7.2 min,phenylhydrazone 7=11.4 min.

The reaction mixture was aged for 30 min at 0-2° C., and TFA was addedmaintaining the temperature between 2 and 7° C. The reaction mixture waswarmed to 50° C. over 30 min, and maintained for 17 h. The nitrogensparge through the reaction mixture was stopped and a slow stream ofnitrogen was maintained over the reaction mixture. During the first hourat 5° C. the color gradually darkened to a deep green, and a relativelysmall amount of a white crystalline precipitate (ammoniumtrifluoroacetate) formed. After 17 h HPLC analysis (same conditions asabove) indicated that the reaction mixture contained 91.6 area %indolenine 8 and 1.5% of unreacted phenylhydrazone remained. Aging themixture for longer periods of time did not increase the assay yield ofindolenine 8.

The reaction mixture was cooled to 12° C., and 7.0 L of MeOH was added.NaBH₄ was added in small (<20 g) portions maintaining the temperaturebelow 15° C. The addition took 30 min. Moderate hydrogen evolution wasobserved during the addition, but it was easily controlled and there wasvirtually no frothing. Near the end of the addition the color rapidlychanged from green to brown and then bright orange. A small amount (<200mL) of a heavier phase had separated (presumably aqueous salts). HPLCanalysis (conditions as before) indicated that all of the indolenine 8had been consumed (90.4 area % CBZ-indoline 9); retention times:indolenine 8=7.5 min, indoline 9=8.2 min. TLC: ethyl ether as solvent,ceric sulfate-ammonium molybdate stain or 1% anisaldehyde stain;retention factors: indolenine 8=0.18, CBZ-indoline 9=0.33.

The color change from green to orange corresponds very closely toreaction end point. The quantity of NaBH₄ required to complete thereaction is heavily dependent on the temperature and rate of addition ofNaBH₄, but the yield and quality of the product is virtually unaffectedprovided that the reaction is complete. The reaction mixture was cooledto 5° C. over a period of 30 min. Then 8 L of 3% aqueous NH₄ OH (8 L)were added to bring the pH of the aqueous phase to 7.4, the mixture wasagitated, and allowed to settle. The temperature rose to 15° C. Thecloudy yellow lower aqueous phase was separated. The organic phase waswashed with 4 L of 3% aqueous NH₄ OH, 2×4 L of water, and 2×4 L ofbrine. The weight of the organic phase after the washings was 53.5 kg,and the assay yield was 94%.

The washed toluene solution was combined with the washed organic phasesof two other similarly processed reactions. The total aldehyde used inthe three reactions was 5.06 kg, (20.5 mol). The total weight ofCBZ-indoline 9 assayed in the combined organic phases was 5.91 kg, (18.3mol, 90% assay yield). The combined organic phases were dried with 5 kgof sodium sulfate, treated with 250 g of Darco G60 carbon for 30 min,and filtered through Solka-floc™. The filtrates were vacuum concentratedat 10 mbar at <25° C. until the residue was near dryness. The solventswitch was completed by slowly bleeding in 30 L of IPAC andreconcentrating to 14 L at 200 mbar at 50-60° C. The mixture was heatedto reflux in order to obtain a clear homogeneous deep orange solution. ¹H NMR analysis indicated that the solution contained ca. 6 mol % ofresidual toluene after solvent switch.

The solution was cooled to 68° C. and seeded with 4 g of crystallineCBZ-indoline 9. The solution was allowed to gradually cool to 26° C.over 6 h and aged for 9 h at 20-26° C. The slurry was cooled to 2° C.over 1 h and aged at 2° C. for 1 h. The product was isolated byfiltration, and the filter cake was washed with 2×2 L of 5° C. IPAC and2×2 L of 5° C. MTBE. The product was dried in the vacuum oven at 30° C.under a nitrogen bleed to give 4.37 kg (74%) of the title compound 9 asa light tan crystalline powder. HPLC analysis of the product indicated99.5 area % purity. The mother liquor (11 L) and the washes contained1.15 kg (19%) of additional product 9 and ca 3% of Cbz-isonipecotic acidphenylhydrazide (retention time=4.8 min).

EXAMPLE 8 ##STR7## CBZ-Spiroindoline-methanesulfonamide (1)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-Spiroindoline (9)                                                                              1.69 kg (5.23 mol)                                       Methanesulfonyl chloride                                                                           599 g (5.23 mol)                                         Et.sub.3 N (KF = 151)                                                                              635 g (6.27 mol)                                         THF (KF = 41)        12 L                                                     ______________________________________                                    

A 22 L flask was charged with the solid CBZ-spiroindoline 9 and then11.5 L of THF and the Et₃ N were transferred into the flask through a10μ inline filter. The resulting homogenous solution was cooled to 0° C.A 1 L dropping funnel was charged with the methanesulfonyl chloride and500 mL of THF. The solution of the MsCl in THF was added to the reactionmixture maintaining the temperature between 0 and 4° C. The additiontook 5 h and was exothermic. A white precipitate, presumablytriethylammonium hydrochloride formed during the addition. HPLC analysisindicated that the reaction was complete at the end of the addition (9was undetectable.

HPLC conditions: 25 cm Dupont Zorbax RXC8 column with 1.5 mL/min flowand detection at 254 nm. Gradient Schedule:

    ______________________________________                                        Time (min)   0.1% aq. H.sub.3 PO.sub.4 :MeCN                                  ______________________________________                                        0            70:30                                                            3            70:30                                                            12           20:80                                                            25           20:80                                                            ______________________________________                                    

Retention times: 9=7.6 min, 1=13.6 min.

After the addition was complete the reaction mixture was warmed to 18°C. and aged for 16 h. There was no change in the appearance of thereaction mixture, and HPLC profile between the end of the addition andafter the 16 h age. The reaction mixture was slowly transferred over 1 hinto a vigorously stirred solution of 30 L of water and 200 mL of 37%aqueous HCl in a 50 L flask. The temperature in the 50 L flask rose from22 to 28° C. The product separated as a pale tan gummy solid whichchanged to a granular solid. The aqueous suspension was cooled to 22° C.and aged for 1 h. The suspension was filtered, and the filter cake waswashed with 2×4 L of MeOH/water (50/50). HPLC analysis indicated that<0.1% of the CBZ-Spiroindoline-methanesulfonamide1 was in the motherliquors.

The filter cake was washed with 4 L of MeOH/water (50/150) to which 50mL of 28% aqueous NH₄ OH had been added. The filter cake was washed with2×4 L of MeOH/water (50/50), and the solid was dried in the vacuum ovenat 50° C. under a nitrogen bleed to give 2.03 kg (97%) of the titleproduct 1 as an off-white powder. HPLC analysis of the solids indicated93.7 area % 1.

EXAMPLE 9 ##STR8## Optional Procedure for Isolation of IntermediateCBZ-Spiroindolenine (8)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Piperidine-4-carboxaldehyde-1-benzyl                                                              12.37 g (0.050 mol)                                       carbamate (5)                                                                 Phenylhydrazine     5.41 g (0.050 mol)                                        Trifluoroacetic acid (TFA)                                                                        11.56 mL, 17.10 g                                                             (0.150 mol)                                               Methylene chloride  500 mL                                                    ______________________________________                                    

The CBZ-aldehyde 5 was dissolved in dichloromethane in a 1 L flaskequipped with Teflon coated magnetic stirring bar. The resultingsolution was cooled to 0° C. Phenylhydrazine was added via a weighedsyringe over 5 min and the temperature was maintained at -1 to 3° C.while nitrogen was continuously bubbled through the reaction mixture.TLC and HPLC analysis indicated complete consumption of the CBZ-aldehyde5 and the appearance of a slight excess (<2%) of phenylhydrazine. TLCconditions: Silica, E. Merck Kieselgel G60 F254 0.25 mm; diethylether/pentane (4/1); and developing agent 0.5% ceric sulfate, 14%ammonium molybdate in 10% aqueous sulfuric acid then heat; Rf: aldehyde5=0.52, phenylhydrazone 7=0.61, phenylhydrazine 6=0.21. HPLC conditions:25 cm Dupont Zorbax RXC8 column at 30° C. with 1.0 mL/min flow anddetection at 254 nm; gradient schedule:

    ______________________________________                                        Time (min)    acetonitrile:water                                              ______________________________________                                        0             57:43                                                           10            65:35                                                           15            75:25                                                           18            75:25                                                           ______________________________________                                    

retention times: phenylhydrazine 6=4.5 min, toluene=7.2 min.phenylhydrazone 7=11.4 min.

The reaction mixture was aged for 10 min at 0-2° C., and TFA was addedby syringe maintaining the temperature between 2 and 7° C. The reactionmixture was warmed to 35° C. over 30 min, and maintained for 17 h. Thenitrogen sparge through the reaction mixture was stopped and a slowstream of nitrogen was maintained over the reaction mixture. During thefirst hour at 35° C. the color gradually darkened to a rosy pink then toa deep green, and a relatively small amount of a white crystallineprecipitate (ammonium trifluoroacetate) formed. After aging for 17 hHPLC analysis (same conditions as above) indicated that the reactionmixture contained 93 area % indolenine 8 and <0.5% of unreactedphenylhydrazone remained. Aging the mixture for longer periods of timedid not increase the assay yield of indolenine 8. The reaction mixturewas cooled to 10° C., and a mixture containing 60 mL 28-30% ammoniumhydroxide, 90 mL water and 150 g crushed ice was added with goodstirring. The color of the mixture changed to a salmon color. Theorganic phase was separated and washed twice with 400 mL water then 100mL saturated aqueous NaCl. The organic phase was dried over magnesiumsulfate and filtered through a plug of 5 g of silica. The filtrate wasevaporated to give 15.84 g (99%) of indolenine 8 as a pale orange oil.

EXAMPLE 10 ##STR9## Procedure for the Preparation ofCBZ-Spiroindoline-methanesulfonamide (1) without Isolation ofIntermediate CBZ-Spiroindoline (9)

Step 1: CBZ-Spiroindoline (9)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Piperidine-4-carboxaldehyde-1-benzyl                                                              49.5 g (0.20 mol)                                         carbamate (5)                                                                 Phenylhydrazine (Aldrich)                                                                         23.7 g (0.22 mol)                                         Trifluoroacetic acid (TFA)                                                                        75.4 g (0.66 mol)                                         Toluene (KF <250 mg/L)                                                                            654 mL                                                    MeCN (KF <250 mg/L) 13.3 mL                                                   NaBH.sub.4          11.3 g, (0.30 mol)                                        Toluene             20 mL                                                     MeOH                50 mL                                                     ______________________________________                                    

A 2% (by volume) solution of MeCN in toluene was made up using 654 mL oftoluene and 13.3 mL of MeCN. In a 2 L 3 neck flask equipped with amechanical stirrer 617 ml of the above solution were degassed by passinga fine stream of nitrogen through the solution for 5 min.Phenylhydrazine and TFA were added to the mixture while still degassing.

The CBZ-aldehyde 5 was dissolved in the rest of the solution preparedabove (50 mL) and degassed by bubbling nitrogen through the solutionwhile in the addition funnel. The solution in the flask was heated to35° C., and the aldehyde solution was slowly added to thephenylhydrazine-TFA over 2 h. The mixture was aged at 35° C. for 16 h.

HPLC conditions: 25 cm Dupont Zorbax RXC8 column at 50° C. with 1 mL/minflow and detection at 220 nm; isocratic 55% MeCN, 45% of 0.1% aqueous H₃PO₄. Typical HPLC profile after 16 h age:

    ______________________________________                                        Retention time (min)                                                                         Area %   Identity                                              ______________________________________                                        1.6            0.1-0.5  phenylhydrazine 6                                     4.1            <0.1     dimer 21                                              4.7            <0.1     aldehyde 5                                            5.0            NA       spiroindoline 9                                       6.3            NA       toluene                                               6.9            97       spiroindolenine 8                                     10.3           <0.2     phenylhydrazone 7                                                    2-3 tot. other impurities <0.2% ea.                            ______________________________________                                    

The mixture was cooled to -10° C. and MeOH was added. A suspension ofsodium borohydride in 20 mL toluene was added in small portions (1 mL)over 30 min taking care that the temperature did not exceed -2° C.

    ______________________________________                                        Area %          Identity                                                      ______________________________________                                        0.1-1           phenylhydrazine 6                                             85-90           CBZ-spiroindoline 9                                           <0.1            CBZ-spiroindolenine 8                                         10-15 tot.      other impurities (<3% ea.)                                    ______________________________________                                    

The temperature was raised to 10° C. over 1 h, and 6% aqueous ammonia(200 mL) was added. The mixture was agitated for 10 min, allowed tosettle for another 10 min, and the lower aqueous phase was drawn off.Acetonitrile (20 mL) and MeOH (20 mL) were added to the organic phaseand it was washed with 150 mL of 15% brine. The organic phase was foundto contain a 92% assay yield of CBZ-spiroindoline 9.

Step 2: CBZ-Spiroindoline-methanesulfonamide (1)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-Spiroindoline (9) (MW = 322.51)                                                                  (0.184 mol)                                            Methanesulfonyl chloride                                                                             21.1 g (0.18.4 mol)                                    DIEA (KF = 150 mg/L)   29.7 g, 40.1 mL                                                               (0.230 mol)                                            THF (KF = 41 mg/L)     150 mL                                                 ______________________________________                                    

The crude solution of CBZ-spiroindoline 9 solution from Step 1 above wasconcentrated in a 1 L 3 neck flask (60-70° C., 150-200 Torr) until 250 gof residue remained. The THF and DIEA were added, and the resultinghomogenous solution was cooled to 0° C. A 125 mL dropping funnel wascharged with the methanesulfonyl chloride and 50 mL of THF. The solutionof MsCl in THF was added over 2 h to the reaction mixture maintainingthe temperature between 0 and 4° C. and the mixture was aged for 2 h at5-8° C. The addition was slightly exothermic. A white precipitate,presumably DIEA-hydrochloride, formed during the addition. HPLCconditions were the same as above. HPLC analysis indicated that thereaction was complete 1 h after the end of the addition (9 wasundetectable) and the assay yield was 94% from 9. Retention time: 1=7.8min. Typical HPLC profile of reaction mixture after 2 h age:

    ______________________________________                                        Area %          Identity                                                      ______________________________________                                        <0.1            CBZ-spiroindoline 9                                           90-92           CBZ-sulfonamide 1                                             8-10 tot.       other impurities (<2% ea.)                                    ______________________________________                                    

The mixture was warmed to 20° C., and 200 mL of 1M aqueous HCl wasadded. The mixture was warmed to 50° C., and the aqueous phase wasseparated. The organic phase was washed sequentialy with 100 mL water,100 mL 5% aqueous sodium bicarbonate, and 100 mL water. The organicphase was transferred to a 1 L 3 neck flask equipped for mechanicalstirring and distillation. The mixture (ca 400 mL) was distilled atatmospheric pressure until 150 mL of distillate had been collected. Thehead temperature reached 107° C.; the pot temperature was 110° C. Thedistillation was continued with continuous addition of n-propanol atsuch a rate as to maintain a constant volume (ca 350 mL) in the pot. Thedistillation was stopped when a total of 525 mL of n-PrOH had been addedand a total of 800 mL of distillate had been collected.

The temperature of both the head and pot rose from 94° C. to 98° C.during the solvent switch. Toluene and n-PrOH form an azeotrope boilingat 97.2° C. composed of 47.5% toluene and 52.5% n-PrOH. The mixture wasallowed to cool gradually to 20° C. over 3 h and aged for 12 h. Themother liquor was found to contain 2% toluene and 4 mg/mL ofsulfonamide. The solubility of the sulfonamide in various mixtures oftoluene and n-PrOH has been determined by HPLC assay:

    ______________________________________                                        % toluene in n-PrOH                                                                          solubility of 1 in mg/mL                                       ______________________________________                                        0              2.36                                                           5              3.02                                                           10             4.23                                                           20             7.51                                                           25             10.3                                                           ______________________________________                                    

The crystalline slurry was filtered and washed with 3×100 mL of n-PrOH.The product was dried in a vacuum oven at 50° C. with a nitrogen bleedfor 16 h to furnish 65.5 g (82% from aldehyde 5) of 6 as a tan solidwith 93.5 wt % purity.

Typical HPLC profile of solid:

    ______________________________________                                        Area %         Identity                                                       ______________________________________                                        <0.1           CBZ-spiroindoline 9                                            >99            CBZ-sulfonamide 1                                              <1 tot.        other impurities (<0.2% ea.)                                   ______________________________________                                    

For additional purification, a 40.0 g sample of the n-PrOH crystallizedsulfonamide was dissolved in 134 mL of EtOAc at 60° C. and treated with8.0 g of Darco G-60 carbon for 1 h at 60° C. After the addition of 2.0 gSolkafloc™, the slurry was filtered through a pad of 4.0 g Solkafloc™,and the pad was washed with 90 mL of EtOAc at 60° C. Prior to theaddition of the carbon the solution was a brown color. The filtrationproceeded well without plugging to give a golden yellow filtrate. Thefiltrate was distilled at atmospheric pressure in a 500 mL flask (pottemperature 80-85° C.) until 100 g (100 mL) of residue remained. Thissolution was allowed to cool to 35° C. over 3 h. Over a 1 h period, 116mL of cyclohexane was added with good agitation at 35° C. The mixturewas cooled to 20° C. over 1 h and aged at 20° C. for 12 h. At 35° C.much of the sulfonamide has crystallized out and the mixture was thick.Addition of cyclohexane at 20° C. makes agitation difficult. After theaging period, the supernatant was found to contain 2.5 mg 1/g. Thecrystalline slurry was filtered and the cake was washed with 77 mL of2:1 cyclohexane-EtOAc and 2×77 mL of cyclohexane. The product was driedin a vacuum oven at 50° C. with a nitrogen bleed for 16 h to furnish34.2 g of 1 (MW=400.3) as a white crystalline solid (85% recovery fromcrude 1, 70% from 5 with >99.9 wt % purity).

EXAMPLE 11 ##STR10## HCl Salt of Spiroindoline-methanesulfonamide (1a)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-spiroindoline-methanesulfonamide (1)                                                              941 g (2.35 mol)                                      Pearlman's catalyst 20% Pd(OH).sub.2 /C                                                               188 g                                                 THF                     8 L                                                   MeOH                    7 L                                                   ______________________________________                                    

The catalyst was suspended in 7 L of MeOH and transferred into the 5 galautoclave followed by the solution of 1 in 8 L of THF.

The mixture was hydrogenolyzed at 25° C. at 80 psi of H₂. After 2.5 hthe temperature was raised to 35° C. over 30 min.

HPLC analysis indicated complete consumption ofCbz-spiroindoline-methanesulfonamide. HPLC conditions: 25 cm DupontZorbax RXC8 column with 1.5 mL/min flow and detection at 254 nm.Gradient Schedule:

    ______________________________________                                        Time (min)   0.1% aq. H.sub.3 PO.sub.4 :MeCN                                  ______________________________________                                        0            70:30                                                            3            70:30                                                            12           20:80                                                            25           20:80                                                            ______________________________________                                         retention times: Spiroindoline = 7.6 min,                                     Cbzspiroindoline-methanesulfonamide = 13.6 min.                          

The mixture was purged with nitrogen and the catalyst was removed byfiltration through Solka-floc™ while still warm. The catalyst was washedwith 4 L of THF and 2 L of MeOH. The pale yellow filtrates wereconcentrated to a thick oil at 10 mbar and <25° C. The solvent switchwas completed by slowly bleeding in 15 L of EtOAc and reconcentrating todryness. The residue solidified to a hard off-white mass. MeOH (1.5 L)was added and the mixture was heated to 70° C. to give a homogenoussolution. While the solution was at 70° C., 10.5 L of EtOAc at 20° C.was added. The temperature fell to 40° C., and the mixture remainedhomogenous.

Subsequent experiments suggested that it is more convenient to solventswitch the MeOH-THF filtrates to. MeOH, concentrate to the desiredvolume, and then add the EtOAc. This avoids the solidification of theresidue upon concentration of the EtOAc solution.

Hydrogen chloride diluted with about an equal volume of nitrogen waspassed into the solution. The temperature rose to 60° C. over the courseof 15 min, and a white precipitate of the hydrochloride salt formed.Diluting the HCl with nitrogen only avoids the reaction mixture suckingback and may not be necessary.

The mixture was cooled in an ice bath, and the hydrogen chlorideaddition was continued for 1 h. The temperature gradually fell to 20° C.The suspension was aged for 2 h while the temperature was lowered to10IC. The crystalline product was isolated by filtration, and the filtercake was washed with 3 L of EtOAc. It was dried in the vacuum oven at35° C. to give 1.18 kg (86%) of the title product 1 a as an off-whitecrystalline solid of >99.5 area % purity by HPLC analysis. HPLCconditions: 25 cm Dupont Zorbax RXC8 column with 1.5 mL/min flow anddetection at 230 nm; isocratic 35% MeCN, 65% of 0.1% aqueous ammoniumacetate. Retention time: 1a=5.4 min.

EXAMPLE 12 ##STR11## Spiroindoline-methanesulfonamide (Free base form)(1b)

A 250 mL aliquot of the filtrate from the Cbz-hydrogenolysis containing4.67 g of 1b (free base) was concentrated to ca 10 mL. The residue wasdissolved in 20 mL of EtOAc and the solution was reconcentrated to ca 10mL. This was repeated once more, and 10 mL of EtOAc was added to theresidue. A crystalline precipitate began to form. MTBE (20 mL) was addedin one portion. Additional crystalline solid precipitated, but thesupernatent still contained a substantial quantity of dissolved productwhich did not precipitate on standing. Hexanes (70 mL) were addeddropwise over 2 h to the mixture with vigorous stirring. The slowaddition of the hexanes is neccessary to avoid the oiling out of theamine. The agitated mixture was aged for 1 h and filtered. The filtercake was washed with 20 mL of 1:1 MTBE-hexanes and then with 20 mL ofhexanes. The product was dried under a stream of nitrogen to give 3.86 g(82%) of the free amine of 1b as an off white crystalline solid of >99.5area % purity. HPLC conditions: 25 cm Dupont Zorbax RXC8 column with 1.5mL/min flow and detection at 230 nm; isocratic 35% MeCN, 65% of 0.1%aqueous ammonium acetate. Retention time: 1b=5.4 min.

EXAMPLE 13A ##STR12##

Spiroindoline-methanesulfonamide (Free base form) (1b)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-Spiroindoline-sulfonamide (1)                                                                   833.5 g (2.08 mol)                                      Pd(OH).sub.2 /C (20% weight of Pd(OH).sub.2)                                                        124.5 (15%)                                             THF                   6.5 L                                                   MeOH                  19.5 L                                                  NH.sub.4 OH (conc)    60 mL                                                   ______________________________________                                    

The hydrogenation was run three (3) times due to equipment limitations;this procedure refers to a single run. The CBZ spiroindolinesulfonamide1 was dissolved in THF (6.5 L, KF=53 μg/μL) and then MeOH(KF=18 μg/mL, 4 L) was added followed by addition of the catalyst andthe slurry was transferred to a 5 gal autoclave. The remainder of theMeOH (2.5 L) was used for rinsing. The mixture was heated to 40° C. at50 psi for 24 hours. The catalyst loading and reaction time are afunction of the purity of starting material 1. This material was uniquerequiring ≧15% catalyst and long reaction time. Purer batches ofspiroindoline required only 5% of catalyst and 4-6 hrs reaction time.

Upon completion (<0.1 A % 1 by LC) the mixture was filtered thru SolkaFloc™ and the carbon cake washed with MeOH (13 L) containing NH₄ OH(0.5%, 60 mL). The combined filtrates (assay shows 1587 g ofspiroindoline amine 1b) were concentrated in vacuo and the resultingsolids were partitioned between 40 L (of toluene:THF (3:1) and 0.5N NaOH(18 L). Although the layers separated easily a heavy precipitate couldbe seen in the aqueous layer. The aqueous suspension was thus extractedwith CH₂ Cl₂ (15 L). The aqueous and organic layer separated slowly.Prior to CH₂ Cl₂ addition THF was added to the aqueous layer along withenough NaCl to saturate the layer. However dissolution of the productwas not achieved which necessitated the use of CH₂ Cl₂.

The combined toluene, THF and CH₂ Cl₂ layers were combined andconcentrated in the batch concentrator. The residue was flushed with 7 Lof CH₃ CN. Finally 10 L of CH₃ CN were added and the solution stoodovernight under N₂ atmosphere.

EXAMPLE 13B ##STR13## Spiroindoline-methanesulfonamide (Free base form)(1b)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        CBZ-Spiroindoline-sulfonamide (1)                                                                    3 kg (7.49 mol)                                        Darco G-60             600 g                                                  Ethyl Acetate          36 L                                                   Absolute Ethanol       189 L                                                  10% Pd/C               450 g                                                  Ammonia Solution       500 ml                                                 Solka Floc ™        2.5 kg                                                 Isopropyl Acetate      65 L                                                   ______________________________________                                    

A mixture of CBZ-spiroindoline (1) (1 kg) and Darco G-60 (200 g) inethyl acetate (9 L) was stirred and heated at 60-65° C. under a nitrogenatmosphere for 8 hours. The Darco was removed by filtration at 60-65°C., the solid washed with hot ethyl acetate (3 L) and the filtrate andwashings combined. LC wt/wt assay confirmed negligible loss to theDarco. The ethyl acetate solution was evaporated to dryness in vacuousing a 20 L Buchi apparatus and then flushed with absolute ethanol (2×5L). This material was then slurried in absolute ethanol (8 L) warmed to65-70° C. and placed in the 20 L autoclave. The batch was rinsed in withabsolute ethanol (1 L). A slurry of 10% Palladium on charcoal (75 g,7.5% by weight) in absolute ethanol (750 ml) was then added to theautoclave and rinsed in with a further portion of absolute ethanol (250ml).

The batch was hydrogenated at 65° C. with vigorous stirring under 40 psihydrogen pressure for 3 hours, a second portion of 10% palladium oncharcoal (75 g) was added, the batch was hydrogenated for a further 2hours and then sealed overnight. The batch was transferred (still hot,60-65° C.) to a 20 L Buchi apparatus and degassed in vacuo to removeformic acid by "feeding and bleeding" absolute ethanol (18 L total).

This procedure was repeated twice more and the three batches werecombined in a 10 gallon glass-lined vessel and the combined batch wasdegassed again by the addition and distillation (in vacuo) of absoluteethanol (2×10 L). Solka floc™ (0.5 kg) was added to the batch and rinsedin with ethanol (10 L). An Estrella filter was loaded with Solkafloc™ (2kg) as a slurry in ethanol (20 L). The resulting mixture was warmed to60-65° C. and then transferred at this temperature via heated filterusing pump to two tared stainless-steel bins. The initial vessel, thefilter, the pump and the lines were rinsed with a hot (60-65° C.)mixture of aqueous ammonia (500 ml) in absolute ethanol (25 L). Thefiltrate and washings were combined in the two stainless-steel bins.

The batch was then transferred to a vessel using an in-line filtercontaining a 10 micron cartridge, and then concentrated in vacuo to lowbulk (˜15 L). The ethanol was replaced by isopropyl acetate by the"feeding and bleeding" of 3x batch volumes of isopropyl acetate (45 Ltotal), while maintaining a batch volume of ˜15 L. The solvent switch,when complete, contained <1% residual ethanol by GC. The batch was thendiluted to ˜33 L by the addition of isopropyl acetate (20 L), and thissolution of spiroindoline-amine 1b (1.855 kg by LC analysis) inisopropyl acetate was used for the next stage of the process.

EXAMPLE 14A ##STR14##

Boc-O-Benzylserine Spiroindoline (11)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Spiroindoline-amine (1b)                                                                        1587      g (5.966 moles)                                   Amino acid (10)   1938      g (6.563 moles)                                    ##STR15##                                                                    DCC               1334.5    g (6.563 moles)                                   HOBT              884       g (6.563 moles)                                   CH.sub.3 CN       25        L                                                 0.5N NaOH         18        L                                                 0.5N HCl          18        L                                                 NaHCO.sub.3 sat.  18        L                                                 iPrOAc            28        L                                                 ______________________________________                                    

The spiroindoline-amine1b in CH₃ CN or iPrOAc:H₂ O (25 L) at ambienttemperature under N₂ was treated in sequence with HOBT (884 g; 1.1 eq)as a solid, DCC (1334.5 g, 1.1 eq) as the melt (heating in hot water at60° C. for ca. 1 hr) and finally the amino acid 10 (1938 g) as thesolid. The mixture was stirred for 3 hr upon which time heavyprecipitation of DCU occurred and LC analysis showed ca. 0.5 A % ofamine 1b remaining. IPAc (9 L) was added, the slurry was filteredthrough Solka Floc™ and the cake was washed with IPAc (19 L). Thecombined organic solution was washed in sequence with 0.5N NaOH (18 L),0.5N HCl (18 L) and saturated NaHCO₃ (18 L). A final water wash at thispoint resulted in an emulsion and was thus eliminated.

The organic layer was concentrated in vacuo and the residue wasdissolved in MeOH or EtOH (10 L final volume). Assay yield 3026 gr(89%).

The use of alternative peptide coupling agents such ascarbonyldiimidazole or formation of mixed anhydrides, such as sec-butylcarbonate, gave inferior yields of 11 and/or 14 with a high degree ofepimerization in the case of the former compound. Other peptide couplingreagents were prohibitively expensive.

EXAMPLE 14B ##STR16##

Boc-O-Benzylserine Spiroindoline (11)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Spiroindoline-amine (1b)                                                                             1.855 kg (6.96 mol)                                    Isopropyl acetate      29 L                                                   Dicyclohexylcarbodiimide (DCC)                                                                       1.58 kg (7.65 mol)                                     1-Hydroxybenzotriazole (HOBt)                                                                        1.03 kg (7.62 mol)                                     N-Boc-O-benzyl-D-Serine                                                                              2.26 kg (7.65 mol)                                     1 M Aqueous sodium hydroxide                                                                         26 L                                                   0.5 M Aqueous hydrochloric acid                                                                      26 L                                                   Satd. Aqueous sodium hydrogen carbonate                                                              26 L                                                   Absolute Ethanol       50 L                                                   ______________________________________                                    

Water (20 L) was added to a stirred solution of thespiroindoline-amine1b (1.855 kg) in isopropyl acetate (33 L) in areaction vessel. The following chemicals were then added sequentially atroom temperature under a nitrogen atmosphere: DCC (1.58 kg, 1.1equivs.), HOBt (1.03 kg, 1.1. equivs.) and finallyN-Boc-O-benzyl-D-Serine (2.26 kg, 1.1 equivs.). The reagents were rinsedin with isopropyl acetate (7 L). The batch was stirred at roomtemperature under nitrogen atmosphere for 5 hours when LC showed theratio of product/starting material to be 99.4/0.6. The mixture was thenfiltered through an Estrella filter using cloth and cardboard only andutilizing a pump into another vessel. The sending vessel was rinsed withisopropyl acetate (22 L) and this was used to rinse the filter, the pumpand the lines into the receiving vessel. The 2-phase mixture in thevessel was stirred for 10 minutes and then allowed to settle for 15minutes. The lower aqueous layer was separated off and the organicsolution was left to stand at room temperature overnight.

The next day, the organic solution was washed with 1M aqueous sodiumhydroxide solution (26 L) then 0.5M aqueous hydrochloric acid (26 L) andfinally saturated aqueous sodium hydrogen carbonate (26 L). LC analysisgave an assay yield of 3.787 kg, 93% overall yield from 7.49 moles (3kg) of starting CBZ-spiroindoline (1). The batch was concentrated invacuo (internal temperature=13-15° C. jacket temperature=40° C.,Vacuum=29") to low bulk (˜15 L) and solvent switched to ethanol by"feeding and bleeding" ethanol (50 L) whilst maintaining the volume at˜15 L. GC showed <1% isopropyl acetate remaining. This solution was usedfor the next stage of the process.

EXAMPLE 15A ##STR17##

O-Benzylserine Spiroindoline (free base form) (12)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Boc-O-Benzylserine Spiroindoline (11)                                                                3026 g (5.57 moles)                                    Methane sulfonic acid (MsOH)                                                                         1.16 L (17.9 moles)                                    MeOH                   10 L                                                   iPrOAc                 24 L                                                   0.5 N NaOH             35 L                                                   ______________________________________                                    

The Boc-O-benzylserine spiroindoline 11 in 10 L of MeOH (or EtOH) wastreated with neat MsOH (1.16 L) added over ca. 30-40 min, (initialtemperature 16° C., final temperature 28° C.). The dark red solution wasaged overnight under N₂. The mixture was then pumped into a 100 Lextractor containing 24 L iPrOAc and 35 L 0.5 N NaOH. The pH of theaqueous layer was 7. NaOH (6M) was added until pH ≧10.5. As the pHincreased the color changed from red to yellow. The layers wereseparated and the organic layer (24 L) was shown by NMR to contain 13mole % of MeOH in iPrOAc [5 volume %]. LC assay 2.48 kg.

EXAMPLE 15B ##STR18## O-Benzylserine Spiroindoline (free base form) (12)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Boc-O-Benzylserine Spiroindoline (11)                                                                3.787 kg (6.96 mol)                                    Methanesulphonic acid  2.006 kg (20.87 mol)                                   Isopropyl acetate      38 L                                                   1 M Aqueous sodium hydroxide                                                                         16 L                                                   50% Aqueous sodium hydroxide                                                                         1.6 L                                                  ______________________________________                                    

Methanesulphonic acid (2.006 kg, 1.355 L, ˜3 equivs.) was added to thestirred solution of Boc-O-benzylserine spiroindoline (11) (3.787 kg) inethanol (total volume -15 L) in a reaction vessel. The batch was warmedto 35-40° C. After 7 hours, LC showed the absence of starting materialand the reaction was allowed to cool to room temperature overnight. Thenext day, water (44 L) was added to the batch with stirring. The batchwas cooled to ˜5°, stirred for 30 minutes and then filtered through anin-line filter (loaded with a 10μ cartridge) into a bin. The batch wasthen sucked back into the vessel. A water rinse (10 L) was used to rinsethe vessel and lines into the bin and this was used to then rinse backinto the vessel. Isopropyl acetate (38 L) was added followed by a 1Maqueous sodium hydroxide (16 L). The batch was cooled to 10-15° C., thepH of the lower aqueous layer was confirmed as ˜7 and 50% aqueous sodiumhydroxide solution was added (1.6 L) (pH>10). The batch was stirred at10-15° C. for 25 minutes and then allowed to settle for 10-15 minutes.The lower aqueous layer was separated (78.1 kg). LC assay indicated 28.4g of 12 (0.85% of theory) contained in the aqueous liquors. Volume ofthe organic solution=51 L. LC assay indicated 3.057 kg, 92% overallyield from 3 kg, 7.49 moles of CBZ-spiroindoline sulfonamide (1). Thissolution was used for the next stage.

EXAMPLE 16A ##STR19##

Boc-Aminoisobutyryl O-Benzylserine Spiroindoline (14)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Spiroindoline amine (12)                                                                       2481      g (5.57 moles)                                     amino acid peptide (13)                                                                        1247.1    g (6.16 moles)                                      ##STR20##                                                                    DCC              1266.7    g (6.16 moles)                                     HOBT             827       g (6.16 moles)                                     IPAc             52        L                                                  H.sub.2 O        37        L                                                  0.5N NaOH        36        L                                                  0.5N HCl         36        L                                                  Sat. NaHCO.sub.3 36        L                                                  ______________________________________                                    

The solution of the amine 12 in IPAc was diluted to a total volume of 39L with IPAc and 37 L of H₂ O was added. The biphasic mixture was thentreated in sequence with HOBT (827 g) as a solid, DCC (1266.7 g) as amelt, and amino acid 13 at ambient temperature under nitrogen. Thereaction mixture was stirred for 2 h upon which time LC analysisindicated dissappearance of the starting material 12 (<0.3 A %). Themixture was filtered through Solka Floc™ and the solids were washed with13 L of IPAc. The material may be stored at this point as a biphasicmixture overnight.

The mixture was transferred to a 100 L extractor, the aqueous layer wasseparated and the organic layer was washed successively with 36 L of0.5N NaOH, 0.5N HCl and saturated NaHCO₃. Assay yield 3160 g (81% fromspiroindoline ±5% for volume measurement error). The solution wasconcentrated to a small volume and was flushed with ethanol (2×4 L). Ifdesired, the inermediate compound 14 may be isolated by adding water tocrystalize it out.

The use of alternative peptide coupling agents such ascarbonyldumidazole or formation of mixed anhydrides, such as sec-butylcarbonate, gave inferior yields of 14 with a high degree ofepimerization. Other peptide coupling reagents were prohibitivelyexpensive.

EXAMPLE 16B ##STR21##

Boc-Aminoisobutyryl O-Benzylserine Spiroindoline (14)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Spiroindoline amine (12)                                                                             3.057 kg (6.89 mol)                                    Dicyclohexylcarbodiimide (DCC)                                                                       1.56 kg (7.56 mol)                                     1-Hydroxybenzotriazole (HOBt)                                                                        1.02 kg (7.55 mol)                                     Boc-2-Aminoisobutyric acid (13)                                                                      1.54 kg (7.58 mol)                                     Isopropyl acetate      32 L                                                   1 M Aqueous sodium hydroxide                                                                         38 L                                                   0.5 M Aqueous hydrochloric acid                                                                      38 L                                                   Satd. aqueous sodium hydrogen carbonate                                                              38 L                                                   Absolute ethanol       45 L                                                   ______________________________________                                    

Water (49 L) was added to the stirred solution of the spiroindolineamine 12 (3.057 kg) in isopropyl acetate (total volume ˜51 L) in areaction vessel at room temperature under a nitrogen atmosphere. Thefollowing chemicals were then added sequentially: DCC (156 kg, ˜1.1equivs.), HOBt (1.02 kg, ˜1.1 equivs.) and finally,N-Boc-2-aminoisobutyric acid 13 (1.54 kg, ˜1.1 equivs.). The mixture wasstirred vigorously at room temperature for 2 hours when LC showed thereaction to be complete. The mixture was filtered to to another vesselvia an Estrella filter using a pump. Isopropyl acetate (22 L) was usedto rinse vessel, the filter, the pump and the lines into the receivingvessel. The 2-phase mixture was then stirred for 5 minutes and thelayers were allowed to separate. The lower aqueous layer was separatedwithout incident (weight of aqueous liquors=51.1 kg). The organicsolution was then washed sequentially with 1M aqueous sodium hydroxide(38 L), 0.5M aqueous hydrochloric acid (38 L) and finally, saturatedaqueous sodium hydrogen carbonate (38 L) without incident.

The organic solution was then transferred using a pump via an in-linefilter (containing a 10μ cartridge) to another vessel for the solventswitch to ethanol. The vessel was rinsed with isopropyl acetate (10 L)and this was used to rinse the pump, the filter and the lines into thereceiving vessel. The filtrate and washings were combined. Totalvolume=75 L (by dipstick). LC assay gave 4.395 kg of Boc-aminoisobutyrylO-benzylserine spiroindoline (14), i.e. 93% overall from 7.49 moles ofstarting CBZ-spiroindoline sulfonamide (1).

The batch was concentrated in vacuo to low bulk (-15 L) and theisopropyl acetate switched to ethanol by "feeding and bleeding" absoluteethanol (45 L total). At the end of the solvent switch, GC showed <1%isopropyl acetate remaining. This solution (25 L) containing 4.395 kg of14 was used for the next stage. If desired, the inermediate compound 14may be isolated by adding water to crystalize it out.

EXAMPLE 17A ##STR22##

Aminoisobutyryl O-Benzylserine Spiroindoline (15)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Boc Spiroindoline (14)                                                                             3160 g (5.03 moles)                                      Methanesulfonic acid (MsOH)                                                                        979 mL (15.1 moles)                                      EtOH                 6.2 L                                                    H.sub.2 O            30 L                                                     1N NaOH              11 L                                                     EtOAc                26 L                                                     Darco 60 activated carbon                                                                          1 Kg                                                     ______________________________________                                    

The Boc spiroindoline 14 was dissolved in 6.2 L of EtOH and treated withMsOH (979 mL). The temperature rose from 20 to 30° C. and the reactionwas allowed to proceed overnight. After 12 hours at 20° C. there wasstill 15 A % of starting material left so the mixture was heated to 35°C. for 6 hours. Upon completion (<0.1 A % 14) the reaction was cooled to200° C. and 30 L of H₂ O were added and the solution was filteredthrough a glass funnel with a polypropylene filter to filter offresidual DCU. The mixture was transferred to a 100 L extractor and 26 Lof EtOAc were added. The aqueous layer was basified via addition ofchilled IN NaOH (11 L) and 1 L of 50% NaOH. Addition of ice was requiredto keep the temperature below 14° C. Higher temperatures resulted insignificant emulsion problems.

The organic layer was distilled at 50° C. at ca. 21" of Hg until KF<1000μg/mL. Lower KF's result in more efficient carbon treatments and betterrecovery at the salt formation step. KF's of 160 μg/mL were achieved atthe 700 g scale. The solution was diluted with ethyl acetate to a totalvolume of 31 L (LC assay 2.40 kg). Activated carbon (Darco G-60) wasadded and the mixture was stirred for 24 h. The mixture was filteredthrough Solka Floc™ and the filter cake was washed with ethyl acetate(16 L), assay 2.34 Kg.

EXAMPLE 17B ##STR23##

Aminoisobutyryl O-Benzylserine Spiroindoline (15)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Boc Spiroindoline (14)                                                                             4.395 kg (6.99 mol)                                      Methanesulfonic acid 2.017 kg (20.99 mol)                                     Ethyl acetate        185 L                                                    1 M Aqueous sodium hydroxide                                                                       16 L                                                     50% Aqueous sodium hydroxide                                                                       2.6 L                                                    Darco G-60           900 g                                                    Solka Floc ™      2.5 kg                                                   ______________________________________                                    

Methanesulfonic acid (2.017 kg, 1.36 L, ˜3 equivs.) was added to thestirred solution of the Boc spiroindoline 14 (4.395 kg) in ethanol(total volume ˜25 L) in a reaction vessel at room temperature. The batchwas warmed to 35-40° C., and stirred overnight. On the next day, thebatch contained ˜1.1 A % of starting material and so the reaction wascontinued for a further 4 hours, then LC showed ratio ofproduct/starting material to be 99.6/0.4. The batch was concentrated invacuo to ˜15 L volume and then diluted with water (44 L). The batch wascooled to 5° C., stirred for 30 minutes and then filtered through aSparkler in-line filter (containing a 10μ cartridge) using a pump toanother vessel to remove a small amount of residual DCU.

The vessel, the pump, the filter and the lines were rinsed with water(10 L), and this was added to the vessel. Ethyl acetate (36 L) was addedto the vessel and the stirred mixture was cooled to 10° C. A solution ofcold (5-10° C.) 1M aqueous sodium hydroxide solution (16 L) and cold(5-10° C.) 50% aqueous sodium hydroxide solution (2.6 L) were added at10° C. and the temperature rose to 14° C. The resulting mixture wasstirred for 15 minutes at <14° C. and then the lower aqueous layerseparated off.

The batch was concentrated in vacuo to ˜20 L volume and then a mixtureof ethyl acetate (35 L) and ethanol (5 L) was fed in while maintainingthe volume at ˜20 L. At the end of this distillation the KF was 9160mgml⁻¹. The batch was solvent switched to ethyl acetate by "feeding andbleeding" ethyl acetate (40 L total). At the end of this distillation,KF was 446 mgml⁻¹. The batch was diluted with ethyl acetate (10 L).

Darco G-60 (900 g) was added to the hazy mixture. This was rinsed inwith ethyl acetate (6 L). This mixture was stirred at room temperatureovernight. Next day, Solka Floc™ (0.5 kg) was added to the stirred batchin the vessel and then Solka Floc™ (2.0 kg) was stirred in a littleethyl acetate and loaded into an Estrella filter. The excess solvent waspumped away through a Sparkler in-line filter containing a 10μcartridge. The slurry was transferred from the vessel through a filterusing a pump and then through another filter to 2×40 L stainless steelbins. Visual inspection showed the liquors to be clear and clean. Thevessel was rinsed with ethyl acetate (22 L) and this was used to rinsethrough the route outlined above to the stainless steel cans. Thecontents of both cans was transferred into a reaction vessel and thesolution was mixed thoroughly.

The batch (58 L) had a KF of 2950 mgml⁻¹ and so was redried byconcentrating in vacuo to 20-25 L volume. The batch was diluted to 46 Lvolume (dipstick) by the addition of ethyl acetate (25 L). The KF was363 mgml⁻¹. The batch was diluted to 62 L volume by the addition ofethyl acetate (17 L) and was used for the final stage of the process.

EXAMPLE 18A ##STR24##Spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamideMethanesulfonate (16)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Amine (15)            2340 g (4.43 moles)                                     Methane sulfonic acid (MsOH)                                                                        316 mL (4.88 moles)                                     EtOAc                 60 L                                                    EtOH                  4.8 L                                                   8% EtOH in EtOAc      20 L                                                    ______________________________________                                    

The volume of the solution of 15 from the previous step was adjusted to60 L with ethyl acetate and EtOH (4.8 L) was added. The MsOH (316 mL)was added in 3 L of EtOAc at 45° C. To the deep red homogeneous solutionwas added 496 g of the title compound Form I seed (10% seed based on theweight of the free amine was employed). The temperature rose to ca. 48°C. and the reaction was aged at 52° C. for 1.5 hours. Analysis indicatedcomplete conversion to the title compound (Form I). (At less than 10%seed longer age (>3 hours) was required). The slurry was allowed to coolto 20° C. overnight and was filtered in a centrifuge under N₂. The cakewas washed with 20 L of 8% EtOH in EtOAc. N₂ is essential duringfiltration because the wet crystals are very hygroscopic. The batch wasdried at 35° C. under vacuum to afford 2.7Kg (56% overall yield) of thetitle compound (Form I) (99.9 A % purity; <0.1% enantiomer).

The conversion of Form II to Form I is also accomplished where the saltis formed in EtOAc-EtOH by addition of MsOH as above and the initialsolution of the salt (at 55° C.) is cooled to 45° C. Crystals startappearing at that temperature and the slurry becomes thicker with time.The temperature is then raised to 51° C. and the slurry is agedovernight. Complete conversion to Form I of 16 should be expected. Thisprocedure may also be employed to prepare seed crystals of Form I of 16.

EXAMPLE 18B ##STR25##Spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamideMethanesulfonate (16)

    ______________________________________                                        Materials:                                                                    ______________________________________                                        Amine (15)            3.1 kg (5.86 mol)                                       Methanesulfonic acid  620 g (6.45 mol)                                        Ethyl acetate         37 L                                                    Absolute ethanol      8.7 L                                                   Spiro[3H-indole-3,4'-piperdin]-1'-yl)-                                                              70 g (0.11 mol)                                         carbonyl)-2-(phenylmethyl-oxy)ethyl]-                                         2-amino-2-methylpropanamide                                                   methanesulfonate (Form I)                                                     ______________________________________                                    

Absolute ethanol (6.4 L) was added to the solution of the amine (15)(3.1 kg) in ethyl acetate (total volume ˜62 L) in a reacttion vessel.The batch was warmed to 50° C. and a solution of methanesulfonic acid(620 g, 412 ml, 1.1 equivs.) in ethyl acetate (11 L) was added over ˜5minutes at 50-54° C. The batch was seeded withspiro[3H-indole-3,4'-piperdin]-1'-yl)-carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate (Form I) (70 g) and the resulting slurry was stirredand heated at 55° C. under nitrogen atmosphere overnight.

The next day, the slurry was cooled to 15-20° C., held for 2 hours andthen dropped to the 50 cm polypropylene filter under nitrogenatmosphere. The solid product was washed with a mixture of absoluteethanol (2.3 L) in ethyl acetate (26 L). The white, solid product wasdug off and dried in an Apex oven in vacuo at 35° C. for an appropriatetime (approx. two days). The driedspiro[3H-indole-3,4'-piperdin]-1'-yl)-carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate (3.352 kg) was sieved using a Jackson-Crockatt sieve togive 3.347 kg (including seed, 70 g)} yield=3.277 kg.

HPLC Conditions:

LC Retention times on Zorbax RX-C8 (4.6 mm×25 cm), λ=210 nm, flowrate=1.5 ml/min.

Compound 1: 60:40 CH₃ CN--H₂ O (1% H₃ PO₄) RT=5.0 min

Compound 1b: 35:65 CH₃ CN--H₂ O (0.1 w % NH₄ OAc) RT=6.2 min.

Compound 10: 60:40 CH₃ CN--H₂ O (0.1 H₃ PO₄) RT=2.9 min.

Compound 11: 60:40 CH₃ CN--H₂ O (0.1% H₃ PO₄) RT=5.4 min.

Compound 12: 40:60 CH₃ CN--H₂ O [pH 5.25 NaH₂ PO4 (6.9 g/L of H₂ O)(adjust pH with NaOH)] RT=5.6 min

Compound 14: 60:40% CH₃ CN--H₂ O (0.1% H₃ PO₄) RT=4.65 min

Compound 15: 40:60% CH₃ CN--H₂ O [pH=5.25 NaH₂ PO4 (6.9 g/L of H₂ O)]adjust pH with NaOH)RT=4.9 min

LC Retention times on Zorbax RX-C8 (4.6 mm×25 cm), λ=210 nm, flowrate=1.2 ml/min, column temperature=48° C.

Solvent A=0.05% Phosphoric acid+0.01% Triethylamine in water

Solvent B=Acetonitrile

Gradient system:

    ______________________________________                                        Time             % A    % B                                                   ______________________________________                                        0 min            95     5                                                     35 min           10     90                                                    38 min           95     5                                                     40 min           95     5                                                     ______________________________________                                    

    ______________________________________                                                    Retention time (mins)                                             ______________________________________                                        Compound 1    25.2                                                            Compound 1b   8.5                                                             Compound 10   20.5                                                            Compound 11   26.3                                                            Compound 12   14.8                                                            Compound 14   25.6                                                            Compound 15   15.7                                                            ______________________________________                                    

EXAMPLE 19

Procedure for Manufacturing 1.0 mg Potency Tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

    ______________________________________                                        Ingredient          Per Tablet                                                                             Per 2600 Tablets                                 ______________________________________                                        Active ingredient (N-[1(R)-[(1,2-dihydro-                                                         1.18   mg    3.068 g                                      1-methane-sulfonylspiro[3H-indole-3,4'-                                       piperdin]-1'-yl)carbonyl]-2-(phenyl-                                          methyloxy)-ethyl]-2-amino-2-methyl-                                           propanamide methanesulfonate)                                                 Calcium Phosphate Dibasic                                                                         47.32  mg    123.03                                                                              g                                      Starch Pregelatinized NF 1500                                                                     30.00  mg    78.0  g                                      Microcrystalline Cellulose NF Avicel PH                                                           15.00  mg    39.0  g                                      101                                                                           Magnesium Stearate Impalpable Powder                                                              0.50   mg    1.3   g                                      NF                                                                            Croscarmellose Sodium NF                                                                          12.75  mg    33.15 g                                      Ethanol 95%         7.5    μl 19.5  ml                                     Water purified (Tablet Weight = 100 g)                                                            22.5   μl 58.5  ml                                     ______________________________________                                    

The active ingredient (equivalent to 1.0 mg anhydrous free base pertablet) was mixed with the calcium phosphate dibasic, the starchpregelatinized NF 1000, the microcrystalline cellulose NF, and half ofthe croscarmellose sodium NF in a high shear granulator for 5 minutes.The 25% ethanol/water granulating solution was slowly added to thepowder mixture with the mixer running over a period of about 1.5 minutesthen granulated for about 7 minutes to form granules. The wet granuleswere dried at about 47° C. (range 46 to 48° C.) in a tray dryer or afluid bed dryer for approximately 3.0 hours. The dried granules werethen milled using a Quadro Comill to achieve fine granules. Aftermilling, the remainder of the croscarmellose sodium NFS was added to thefine granules and mixed in a V blender for about 10 minutes. Magnesiumstearate impalpable powder NF was added to this blend through a 60 meshstainless steel screen and blended in the V blender for about 1 minute.The lubricated mixture was compressed to provide tablets of 1.0 mgactive ingredient (free base equivalent).

EXAMPLE 20

Procedure for Manufacturing 1.0 mg Potency Coated Tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

    ______________________________________                                        Ingredient          Per Tablet                                                                             Per 2600 Tablets                                 ______________________________________                                        Hydroxypropyl Methylcellulose USP                                                                 0.80   mg    2.08  g                                      (HPMC)                                                                        Hydroxypropyl Cellulose NF with <0.3%                                                             0.80   mg    2.08  g                                      Silica (HPC)                                                                  Titanium Dioxide USP                                                                              0.32   mg    0.83  g                                      Talc USP Purified   0.08   mg    0.21  g                                      Water Purified (Film Coated Tablet                                                                To 20  μl To 52 ml                                     Weight = 102 g)                                                               ______________________________________                                    

The titanium dioxide and talc, USP were mixed and passed through a 60mesh stainless steel screen. This mixture was mixed with HPMC and HPC toform a dry blend. The dry blend was added to water (20 ml) which waspreviously heated to 90° C. with mild agitation to ensure that the blendis wetted to form a slurry. The remainder of the water (up to 32 ml) wasadded to the slurry at ambient temperature with gentle agitation to forma suspension. The suspension was then applied to the tablets from theprevious Example using the following guidelines to provide the coatedtablets.

Pan: suitable size

Pan Speed: 20 RPM

Nozzles: 2850 liquid/120 air

Inlet Temperature: 85° C.

Bed Temperature: 47° C.

Spray Rate: ca. 2.0 g/minute/kg Tablets

EXAMPLE 21

Procedure for Manufacturing 5.0 mg Potency Tablets ofN-[1(R)[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

    ______________________________________                                        Ingredient        Per Tablet                                                                              Per 25,000 Tablets                                ______________________________________                                        Active Ingredient (N-[1(R)-[1,2-                                                                5.91    mg    147.8  g                                      dihydro-1-methane-sulfonylspiro-                                              [3H-indole-3,4'-piperdin]-1'-yl)-                                             carbonyl]-2-(phenylmethyloxy)-                                                ethyl]-2-amino-2-methylpropan-                                                amide methanesulfonate)                                                       Calcium Phosphate Dibasic                                                                       188.10  mg    4.70   kg                                     Starch Pregelatinized NF 1500                                                                   120.00  mg    3.00   kg                                     Microcrystalline Cellulose NF Avicel                                                            60.00   mg    1.50   kg                                     PH 101                                                                        Magnesium Stearate Impalpable                                                                   2.00    mg    50.0   g                                      Powder NF                                                                     Croscarmellose Sodium NF                                                                        24.00   mg    600    g                                      Ethanol 95%       30      μl 750    ml                                     Water purified (Tablet Weight =                                                                 90      μl 2.25   l                                      400 g)                                                                        ______________________________________                                    

The active ingredient (equivalent to 5.0 mg anhydrous free base pertablet) was mixed with the calcium phosphate dibasic, the starchpregelatinized NF 1000, the microcrystalline cellulose NF, and half ofthe croscarmellose sodium NF in a high Fielder 10/25 mixer for about 6minutes. The 25% ethanol/water granulating solution was slowly added tothe powder mixture with the mixer running over a period of about 1.5minutes then granulated for about 8 minutes to form granules. The wetgranules were dried at about 47° C. (range 46 to 48° C.) in a tray dryeror a fluid bed dryer for approximately 3.0 hours. The dried granuleswere then milled using a Quadro Comill to achieve fine granules. Aftermilling, the remainder of the croscarmellose sodium NFS was added to thefine granules and mixed in a V blender for about 10 minutes. Magnesiumstearate impalpable powder NF was added to this blend through a 60 meshstainless steel screen and blended in the V blender for about 1 minute.The lubricated mixture was compressed to provide tablets of 5.0 mgactive ingredient (free base equivalent).

EXAMPLE 22

Procedure for Manufacturing 5.0 mg Potency Coated Tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

    ______________________________________                                        Ingredient         Per Tablet                                                                             Per 25,000 Tablets                                ______________________________________                                        Hydroxypropyl Methylcellulose USP                                                                3.2    mg    80     g                                      (HPMC)                                                                        Hydroxypropyl Cellulose NF with                                                                  3.2    mg    80.0   g                                      <0.3% Sillca (HPC)                                                            Titanium Dioxide USP                                                                             1.28   mg    32.0   g                                      Talc USP Purified  0.32   mg    8.0    g                                      Water Purified (Film Coated Tablet                                                               To 80  μl To 200 ml                                     Weight = 408 g)                                                               ______________________________________                                    

Using essentially the procedure of the prior Example 20 and applying thesuspension to the tablets from the previous Example, 5.0 mg potencycoated tablets were formed.

EXAMPLE 23

Procedure for Manufacturing 25 mg Potency Tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

    ______________________________________                                        Ingredient        Per Tablet                                                                              Per 25,000 Tablets                                ______________________________________                                        Active Ingredient (N-[1(R)-[(1,2-                                                               29.55   mg    738.75 g                                      dihydro-1-methane-sulfonylspiro-                                              [3H-indole-3,4'-piperdin]-1'-yl)-                                             carbonyl]-2-(phenylmethyloxy)-                                                ethyl]-2-amino-2-methylpropanamide                                            methanesulfonate)                                                             Calcium Phosphate Dibasic                                                                       174.46  mg    4.361  kg                                     Starch Pregelatinized NF 1500                                                                   113.00  mg    2.825  kg                                     Microcrystalline Cellulose NF Avicel                                                            57.00   mg    1.425  kg                                     PH 101                                                                        Magnesium Stearate Impalpable                                                                   2.00    mg    50.0   g                                      Powder NF                                                                     Croscarmellose Sodium NF                                                                        24.00   mg    600    g                                      Ethanol 95%       30      μl 750    ml                                     Water purified (Tablet Weight =                                                                 90      μl 2.25   l                                      400 g)                                                                        ______________________________________                                    

The active ingredient (equivalent to 25 mg anhydrous free base pertablet) was mixed with the calcium phosphate dibasic, the starchpregelatinized NF 1000, the microcrystalline cellulose NF, and half ofthe croscarmellose sodium NF in a high shear granulator Fielder 10/25mixer for about 6 minutes. The 25% ethanol/water granulating solutionwas slowly added to the powder mixture with the mixer running over aperiod of about 1.5 minutes then granulated for about 8 minutes to formgranules. The wet granules were dried at about 47° C. (range 46 to 48°C.) in a tray dryer or a fluid bed dryer for approximately 3.0 hours.The dried granules were then milled using a Quadro Comill to achievefine granules. After milling, the remainder of the croscarmellose sodiumNFS was added to the fine granules and mixed in a V blender for about 10minutes. Magnesium stearate impalpable powder NF was added to this blendthrough a 60 mesh stainless steel screen and blended in the V blenderfor about 1 minute. The lubricated mixture was compressed to providetablets of 25 mg active ingredient (free base equivalent).

EXAMPLE 24

Procedure for Manufacturing 25 mg Potency Coated Tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

    ______________________________________                                        Ingredient         Per Tablet                                                                             Per 25,000 Tablets                                ______________________________________                                        Hydroxypropyl Methylcellulose USP                                                                3.2    mg    80     g                                      (HPMC)                                                                        Hydroxypropyl Cellulose NF with                                                                  3.2    mg    80.0   g                                      <0.3% Silica (HPC)                                                            Titanium Dioxide USP                                                                             1.28   mg    32.0   g                                      Talc USP Purified  0.32   mg    8.0    g                                      Water Purified (Film Coated Tablet                                                               To 80  μl To 200 ml                                     Weight = 408 g)                                                               ______________________________________                                    

Using essentially the procedure of Example 20 and applying thesuspension to the tablets from the previous Example, 25 mg potencycoated tablets were formed.

EXAMPLE 25

Procedure for Manufacturing 100 mg Potency Tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

    ______________________________________                                        Ingredient         Per Tablet                                                                              Per 2600 Tablets                                 ______________________________________                                        Active Ingredient (N-[1(R)-[(1,2-                                                                118.20  mg    307.3 g                                      dihydro-1-methane-sulfonylspiro[3H-                                           indole-3,4'-piperdin]-1'-yl)-carbonyl]-                                       2-(phenylmethoxyloxy)-ethyl]-2-amino-                                         2-methylpropanamide methane-                                                  sulfonate)                                                                    Calcium Phosphate Dibasic                                                                        81.80   mg    212.7 g                                      Starch Pregelatinized NF 1500                                                                    78.00   mg    202.8 g                                      Microcrystalline Cellulose NF Avicel                                                             60.00   mg    156.0 g                                      PH 101                                                                        Magnesium Stearate Impalpable Powder                                                             2.00    mg    5.20  g                                      NF                                                                            Croscarmellose Sodium NF                                                                         60.00   mg    156.0 g                                      Ethanol 95%        30.0    μl 78.0  ml                                     Water purified (Tablet Weight = 400 g)                                                           90.0    μl 234.0 ml                                     ______________________________________                                    

The active ingredient (equivalent to 100 mg anhydrous free base pertablet) was mixed with the calcium phosphate dibasic, the starchpregelatinized NF 1000, the microcrystalline cellulose NF, and half ofthe croscarmellose sodium NF in a high shear granulator for 5 minutes.The 25% ethanol/water granulating solution was slowly added to thepowder mixture with the mixer running over a period of about 1.5 minutesthen granulated for about 7 minutes to form granules. The wet granuleswere dried at about 47° C. (range 46 to 48° C.) in a tray dryer or afluid bed dryer for approximately 3.0 hours. The dried granules werethen milled using a Quadro Comill to achieve fine granules. Aftermilling, the remainder of the croscarmellose sodium NFS was added to thefine granules and mixed in a V blender for about 10 minutes. Magnesiumstearate impalpable powder NF was added to this blend through a 60 meshstainless steel screen and blended in the V blender for about 1 minute.The lubricated mixture was compressed to provide tablets of 100 mgactive ingredient (free base equivalent).

EXAMPLE 26

Procedure for Manufacturing 100 mg Potency Coated Tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

    ______________________________________                                        Ingredient         Per Tablet                                                                              Per 2600 Tablets                                 ______________________________________                                        Hydroxypropyl Methylcellulose USP                                                                3.2     mg    8.32   g                                     (HPMC)                                                                        Hydroxypropyl Cellulose NF with                                                                  3.2     mg    8.32   g                                     <0.3% Silica (HPC)                                                            Titanium Dioxide USP                                                                             1.28    mg    3.33   g                                     Talc USP Purified  0.32    mg    0.83   g                                     Water Purified (Film Coated Tablet                                                               To 80.0 μl To 208 ml                                    Weight = 408 g)                                                               ______________________________________                                    

Using essentially the procedure of Example 20 and applying thesuspension to the tablets from the previous Example, 100 mg potencycoated tablets were formed.

EXAMPLE 27

Preparation of amorphous form ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

As a mimic of the tablet formulation process, a concentrated solution ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate (118 mg) in 120 μL 25% aqueous ethanol (980 mg/ml) wasevaporated at 40° C. to give a solid. The lack of crystallinity wasconfirmed by X-ray analysis. The X-ray diffraction pattern showed anamorphous halo. Examination of the solid under microscopy showed nobiorefringence.

The solid state chemical stability of the amorphous form was studiedafter 12 weeks at 40° C., 60° C. and 80° C., and it was found to exhibitexcellent stability. After 12 weeks at 40° C., 100% of the initialcompound was present; after 12 weeks at 60° C., 99.7% of the initialcompound was present; after 12 weeks at 80° C., 97.8% of the initialcompound was present.

EXAMPLE 28

Chemical Stability of Film Coated Tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate Following 6.0 Months Stability Study

The results of a 6.0 month stability study of film coated tablets ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate are presented below.

    ______________________________________                                                           % of initial                                                                            % of initial                                     Stability Chamber  1.0 mg dose                                                                             100 mg dose                                      ______________________________________                                        30° C./Amient Humidity                                                                    99        99                                               30° C./75% Relative Humidity                                                              99        99                                               40° C./75% Relative Humidity                                                              99        99                                               ______________________________________                                    

In particular, no degradates were observed for the 100 mg dose.Degradates were observed for the 1.0 mg dose varying from 0.1 to 0.7area % relative to active only at 40° C./75% relative humidity.Furthermore, the tablet dissolution, disintegration, and hardness forboth the 1.0 mg dose and the 100 mg dose were satisfactory following 6.0months storage under the above conditions.

EXAMPLE 29

Tablet Crushing Strength and Disintegration Times of 100 mg PotencyTablets of N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate

Tablets of 400 mg compression weight were prepared by the procedures ofthe above examples using 118.2 mg ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate per tablet (29.6% tablet weight). All granulations wereprepared on a several gram scale by pestle and morter using 25%ethanol/75% water as a granulating solution. Tablets were compressed ona Carver Press under 1000 lb force (uncoated tablet cores), unlessotherwise noted.

Formulation A: wet granulation--Starch Pregelatinized NF 1500 (34.8%);Microcrystalline Cellulose NF Avicel PH 101 (34.8%); Talc USP (0.6%);Magnesium Stearate (0.3%).

Formulation B: direct compression--Calcium Phosphate Dibasic (26.3%);Microcrystalline Cellulose NF Avicel PH 102 (39%); Talc USP (1.2%);Magnesium Stearate (0.6%); Croscarmellose Sodium NF (3%).

Formulation C: wet granulation, compressed under 500 lb force--StarchPregelatinized NF 1500 (33.3%); Microcrystalline Cellulose NF Avicel PH101 (33.3%); Talc USP (0.6%); Magnesium Stearate (0.3%); CroscarmelloseSodium NF (3%=1.5% intragranular+1.5% extragranular).

Formulation D: wet granulation--Starch Pregelatinized NF 1500 (33.3%);Microcrystalline Cellulose NF Avicel PH 101 (33.3%); Talc USP (0.6%);Magnesium Stearate (0.3%); Croscarmellose Sodium NF (3% extragranular).

Formulation E: wet granulation--Microcrystalline Cellulose NF Avicel PH101 (36.4%); Calcium Phosphate (26.3%); Talc USP (1.2%); MagnesiumStearate (0.6%); Croscarmellose Sodium NF (6%=3% intragranular+3%extragranular).

Formulation F: wet granulation--Microcrystalline Cellulose NF Avicel PH101 (15.0%); Calcium Phosphate (29.5%); Starch Pregelatinized NF 1500(19.5%); Magnesium Stearate (0.5%); Croscarmellose Sodium NF (6%=3%intragranular+3% extragranular).

Formulation G: wet granulation--Microcrystalline Cellulose NF Avicel PH101 (15.0%); Calcium Phosphate (26.5%); Starch Pregelatinized NF 1500(19.5%); Magnesium Stearate (0.5%); Croscarmellose Sodium NF (9%=3%intragranular+6% extragranular).

Formulation H: wet granulation--Microcrystalline Cellulose NF Avicel PH101 (15.0%); Calcium Phosphate (26.5%); Starch Pregelatinized NF 1500(19.5%); Magnesium Stearate (0.5%); Croscarmellose Sodium NF (9%=3%intragranular+6% extragranular); Super Disintegrant (12% extragranular).

    ______________________________________                                        Formulation                                                                            Tablet Hardness (kP)                                                                          Disintegration Time (min)                            ______________________________________                                        A        Does not break  13.0                                                 B        23.3 (one tablet)                                                                             7.0                                                  C        23.7 (one tablet)                                                                             14.0                                                 D        29.2 ± 1.2 (three tablets)                                                                  13.5 ± 0.25                                      E        14.1 ± 0.7 (three tablets)                                                                 12.5 ± 1.6                                        F        13.5 ± 1.1 (three tablets)                                                                 20.5 ± 0.0                                        G        18.5 (two tablets)                                                                            14.0                                                 H        14.9 (two tablets)                                                                            7.5                                                  ______________________________________                                    

As demonstrated above, the present formulations have superior propertiesregarding strength and stability. The tablet hardness is suitable forfilm coating and disintegration time is not too long.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.For example, effective dosages other than the particular dosages as setforth herein above may be applicable as a consequence of variations inthe responsiveness of the mammal being treated for any of theindications with the compound of the invention indicated above.Likewise, the specific pharmacological responses observed may varyaccording to and depending upon the particular active compound selectedor whether there are present pharmaceutical carriers, as well as thetype of formulation and mode of administration employed, and suchexpected variations or differences in the results are contemplated inaccordance with the objects and practices of the present invention. Itis intended, therefore, that the invention be defined by the scope ofthe claims which follow and that such claims be interpreted as broadlyas is reasonable.

What is claimed is:
 1. A pharmaceutical composition comprising:0.1 to50% by weight of an active ingredientN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methyl-propanamide,or a pharmaceutically acceptable salt thereof; 20 to 40% by weight of abinder/diluent which is selected from: hydroxypropyl methylcellulose,hydroxypropyl cellulose, pregelatinized starch and polyvinylpyrrolidone;10 to 20% by weight of a first diluent which is selected from: lactose,microcrystalline cellulose, calcium phosphate dibasic, mannitol,powdered cellulose and pregelatinized starch; 20 to 50% by weight of asecond diluent which is selected from: lactose, microcrystallinecellulose, calcium phosphate dibasic, mannitol, powdered cellulose andpregelatinized starch; 5 to 15% by weight of a disintegrant which isselected from microcrystalline or croscarmellose sodium; and 0.05 to 5%by weight of a lubricant which is selected from magnesium stearate,calcium stearate, and steric acid; wherein sum of the above proportionsof the active ingredient, the binder/diluent, the first diluent, thesecond diluent, the disintegrant, and the lubricant are not greater than100% by weight.
 2. A pharmaceutical composition comprising:about 1 to30% by weight of an active ingredientN-[1-(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methyl-propanamide,or a pharmaceutically acceptable salt thereof; about 20 to 40% by weightof pregelatinized starch; about 10 to 20% by weight of microcrystallinecellulose; about 20 to 50% by weight of calcium phosphate dibasic; about5 to 15% by weight of croscarmellose sodium; and about 0.05 to 5% byweight of magnesium stearate,wherein the sum of the above proportions ofactive ingredient, pregelatinized starch, microcrystalline cellulose,calcium phosphate dibasic, croscarmellose sodium, and magnesium stearateare not greater than 100% by weight.
 3. The pharmaceutical compositionof claim 2 comprising: about 1 to 2% by weight of the active ingredient;about 25 to 35% by weight of pregelatinized starch; about 10 to 20% byweight of microcrystalline cellulose; about 45 to 55% by weight ofcalcium phosphate dibasic; about 4 to 8% by weight of croscarmellosesodium; and about 0.1 to 1% by weight of magnesium stearate wherein thesum of the above proportions of active ingredient, pregelatinizedstarch, microcrystalline cellulose, calcium phosphate dibasic,croscarmellose sodium, and magnesium stearate are not greater than 100%by weight.
 4. The pharmaceutical composition of claim 3 comprising:about 1.18% by weight of the active ingredient which isN-[1(R)-[(1,2-dihydro-1-methane-sulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate; about 30.0% by weight of pregelatinized starch; about15.0% by weight of microcrystalline cellulose; about 47.3% by weight ofcalcium phosphate dibasic; about 6.0% by weight of croscarmellosesodium; and about 0.5% by weight of magnesium stearate.
 5. Thepharmaceutical composition of claim 4 further comprising a coating ofabout 0.8% by weight of hydroxypropyl methylcellulose; about 0.8% byweight of hydroxypropyl cellulose; about 0.32% by weight of titaniumdioxide; and about 0.08% by weight of talc (as a percentage of the coretablet weight).
 6. The pharmaceutical composition of claim 3 comprising:about 1.48% by weight of the active ingredient which isN-[1(R)-[(1,2-dihydro-1-methane-sulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate; about 30.0% by weight of pregelatinized starch; about15.0% by weight of microcrystalline cellulose; about 47.0% by weight ofcalcium phosphate dibasic; about 6.0% by weight of croscarmellosesodium; and about 0.5% by weight of magnesium stearate.
 7. Thepharmaceutical composition of claim 6 further comprising a coating ofabout 0.8% by weight of hydroxypropyl methylcellulose; about 0.8% byweight of hydroxypropyl cellulose; about 0.32% by weight of titaniumdioxide; and about 0.08% by weight of talc (as a percentage of the coretablet weight).
 8. The pharmaceutical composition of claim 2 comprising:about 5 to 10% by weight of the active ingredient, about 25 to 35% byweight of pregelatinized starch; about 10 to 20% by weight ofmicrocrystalline cellulose; about 40 to 50% by weight of calciumphosphate dibasic; about 4 to 8% by weight of croscarmellose sodium; andabout 0.1 to 1% by weight of magnesium stearate, wherein the sum of theabove proportions of the active ingredient, pregelatinized starch,microcrystalline cellulose, calcium phosphate dibasic, croscarmellosesodium, and magnesium stearate are not greater than 100% by weight. 9.The pharmaceutical composition of claim 8 comprising: about 7.39% byweight of the active ingredient which isN-[1(R)-[(1,2-dihydro-1-methane-sulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate; about 28.2% by weight of pregelatinized starch; about14.2% by weight of microcrystalline cellulose; about 43.6% by weight ofcalcium phosphate dibasic; about 6.0% by weight of croscarmellosesodium; and about 0.5% by weight of magnesium stearate.
 10. Thepharmaceutical composition of claim 9 further comprising a coating ofabout 0.8% by weight of hydroxypropyl methylcellulose; about 0.8% byweight of hydroxypropyl cellulose; about 0.32% by weight of titaniumdioxide; and about 0.08% by weight of talc (as a percentage of the coretablet weight).
 11. The pharmaceutical composition of claim 2comprising: about 25 to 35% by weight of the active ingredient, about 15to 25% by weight of pregelatinized starch; about 10 to 20% by weight ofmicrocrystalline cellulose; about 15-25% by weight of calcium phosphatedibasic; about 10 to 20% by weight of croscarmellose sodium; and about0.1 to 1% by weight of magnesium stearate, wherein the sum of the aboveproportions of the active ingredient, pregelatinized starch,microcrystalline cellulose, calcium phosphate dibasic, croscarmellosesodium, and magnesium stearate are not greater than 100% by weight. 12.The pharmaceutical composition of claim 11 comprising: about 29.5% byweight of the active ingredient which isN-[1(R)-[(1,2-dihydro-1-methane-sulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate; about 19.5% by weight of pregelatinized starch; about15.0% by weight of microcrystalline cellulose; about 20.4% by weight ofcalcium phosphate dibasic; about 15.0% by weight of croscarmellosesodium; and about 0.5% by weight of magnesium stearate.
 13. Thepharmaceutical composition of claim 12 further comprising a coating ofabout 0.8% by weight of hydroxypropyl methylcellulose; about 0.8% byweight of hydroxypropyl cellulose; about 0.32% by weight of titaniumdioxide; and about 0.08% by weight of talc (as a percentage of the coretablet weight).
 14. A process for the preparation of a tablet containingan active ingredient of the compound:N-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide,or a pharmaceutically acceptable salt thereof, comprising the stepsof:(1) forming a powder blend of the active ingredient with abinder/diluent, a first diluent, a second diluent, and a first portionof a disintegrant, (2) wet granulating the powder blend with a solutionof ethanol/water to form granules, (3) drying the granules to remove theethanol/water, (4) adding a second portion of a disintegrant; (5)lubricating the granules; and (6) compressing the dried granules into adesired tablet form.
 15. The process of claim 14 wherein the activeingredient isN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate.
 16. The process of claim 14 further comprising coatingthe tablet by:(1) dry blending titanium dioxide with hydroxypropylmethylcellulose and hydroxypropyl cellulose to form a dry powderedblend; (2) adding the dry powdered blend to water to form a slurry; (3)adding water to the slurry with stirring to form a suspension; and (4)applying the suspension to the tablets.
 17. The process of claim 14,comprising the steps of:(1) forming a powder blend of the activeingredient with a binder/diluent, a first diluent, a second diluent, anda disintegrant, from 2 to 25 minutes using a mixer; (2) wet granulatingthe powder blend by adding a solution of ethanol/water to the powderblend while mixing over a 1 to 30 minute period to form granules; (3)drying the granules to remove water with heated air in a fluid bed dryeror tray dryer for 10 minutes to 24 hours; (4) milling the dried granulesto a uniform size; (5) adding and blending a disintegrant with the driedmilled particles for 2 to 30 minutes; (6) adding and blending alubricant to the mixture containing the disintegrant for 30 seconds to20 minutes; and (7) compressing the lubricated granules mixture into adesired tablet form.
 18. The process of claim 17 wherein the activeingredient isN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate.
 19. The process of claim 18 further comprising coatingthe tablet by:(1) dry blending titanium dioxide with hydroxypropylmethylcellulose and hydroxypropyl cellulose to form a dry powderedblend; (2) adding the dry powdered blend to water to form a slurry; (3)adding water to the slurry with stirring to form a suspension; and (4)applying the suspension to the tablets.
 20. The process of claim 18wherein the binder/diluent is pregelatinized starch; the first diluentis microcrystalline cellulose; the second diluent is calcium phosphatedibasic; the disintegrant is croscarmellose sodium; and the lubricant ismagnesium stearate.
 21. The process of claim 14, comprising the stepsof:(1) forming a powder blend of the active ingredient withpregelatinized starch, microcrystalline cellulose, calcium phosphatedibasic, and croscarmellose sodium, in a mixer for about 3 to 25minutes; (2) wet granulating the powder blend by adding a solution of25% ethanol/75% water (w/w) to the powder blend while mixing over a 1 to30 minute period to form granules; (3) drying the granules on a traydryer or a fluid bed dryer for about 1 to 12 hours to remove theethanol/water; (4) milling the dried granules to a uniform size using aQuadro Comill or Fitz type mill; (5) adding and blending croscarmellosesodium with the dried milled particles for about 5 to 30 minutes; (6)adding and blending magnesium stearate to the mixture containing thecroscarmellose sodium with a V blender for about 1 to 5 minutes; and (7)compressing the lubricated granules mixture into a desired tablet form.22. The process of claim 21 wherein the active ingredient isN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate.
 23. A solid dosage form containing an activeingredient ofN-[1(R)-[(1,2-dihydro-1-methanesulfonylspiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide,or a pharmaceutically acceptable salt thereof, wherein the dosage formis prepared by the process of claim
 14. 24. An amorphous form of thecompoundN-[1(R)-[(1,2-dihydro-1-methane-sulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenyl-methyloxy)ethyl]-2-amino-2-methylpropanamidemethanesulfonate.
 25. The amorphous form of claim 24 characterized by anX-ray diffraction pattern exhibiting an amorphous halo.
 26. Theamorphous form of claim 24 characterized by showing no biorefringenceupon examination under microscopy.
 27. An amorphous form ofN-[1(R)-[(1,2-dihydro-1-methanesulfonyl-spiro[3H-indole-3,4'-piperdin]-1'-yl)carbonyl]-2-(phenylmethyl-oxy)ethyl]-2-amino-2-methyl-propanamidemethanesulfonate which is produced by the process of claim
 14. 28. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and an effective amount of the amorphous form of claim 24.